Aminimide-containing molecules and materials as molecular recognition agents

ABSTRACT

The design and synthesis of novel aminimide-based molecular modules and the use of the modules in the construction of new molecules and fabricated materials is disclosed. The new molecules and fabricated materials are molecular recognition agents useful in the design and synthesis of drugs, and have applications in separations and materials science.

This is a continuation of application Ser. No. 08/765,173, filed Feb.16, 1996, now U.S. Pat. No. 5,981,467, which is a continuation ofapplication Ser. No. 08/204,206, filed Mar. 27, 1995, now U.S. Pat. No.5,705,585, which is a continuation-in-part of each of abandonedapplications Ser. Nos. 07/906,769, filed Jun. 30, 1992, 07/906,770,filed June 30, 1992 and 08/041,559, filed Apr. 2, 1993.

FIELD OF THE INVENTION

The present invention relates to the logical development of biochemicaland biopharmaceutical agents and of new materials including fabricatedmaterials such as fibers, beads, films, and gels. Specifically, theinvention relates to the development of molecular modules based onaminimide and related structures, and to the use of these modules in theassembly of molecules and fabricated materials with tailored properties,which are determined by the contributions of the individual buildingmodules. The molecular modules of the invention are preferably chiral,and can be used to synthesize new compounds and fabricated materialswhich are able to recognize biological receptors, enzymes, geneticmaterials, and other chiral molecules, and are thus of great interest inthe fields of biopharmaceuticals, separation and materials science.

BACKGROUND OF THE INVENTION

The discovery of new molecules has traditionally focused in two broadareas, biologically active molecules, which are used as drugs for thetreatment of life-threatening diseases, and new materials, which areused in commercial, especially high-technological applications. In bothareas, the strategy used to discover new molecules has involved twobasic operations: (i) a more or less random choice of a molecularcandidate, prepared either via chemical synthesis or isolated fromnatural sources, and (ii) the testing of the molecular candidate for theproperty or properties of interest. This discovery cycle is repeatedindefinitely until a molecule possessing the desirable properties islocated. In the majority of cases, the molecular types chosen fortesting have belonged to rather narrowly defined chemical classes. Forexample, the discovery of new peptide hormones has involved work withpeptides; the discovery of new therapeutic steroids has involved workwith the steroid nucleus; the discovery of new surfaces to be used inthe construction of computer chips or sensors has involved work withinorganic materials, etc. As a result, the discovery of new functionalmolecules, being ad hoc in nature and relying predominantly onserendipity, has been an extremely time-consuming, laborious,unpredictable, and costly enterprise.

A brief account of the strategies and tactics used in the discovery ofnew-molecules is described below. The emphasis is on biologicallyinteresting molecules; however, the technical problems encountered inthe discovery of biologically active molecules as outlined here are alsoillustrative of the problems encountered in the discovery of moleculeswhich can serve as new materials for high technological applications.Furthermore, as discussed below, these problems are also illustrative ofthe problems encountered in the development of fabricated materials forhigh technological applications.

2.1 Drug Design

Modern theories of biological activity state that biological activities,and therefore physiological states, are the result of molecularrecognition events. For example, nucleotides can form complementary basepairs so that complementary single-stranded molecules hybridizeresulting in double- or triple-helical structures that appear to beinvolved in regulation of gene expression. In another example, abiologically active molecule, referred to as a ligand, binds withanother molecule, usually a macromolecule referred to as ligand-acceptor(e.g. a receptor or an enzyme), and this binding elicits a chain ofmolecular events which ultimately gives rise to a physiological state,e.g. normal cell growth and differentiation, abnormal cell growthleading to carcinogenesis, blood-pressure regulation,nerve-impulse-generation and -propagation, etc. The binding betweenligand and ligand-acceptor is geometrically characteristic andextraordinarily specific, involving appropriate three-dimensionalstructural arrangements and chemical interactions.

2.1.1 Design and Synthesis of Nucleotides

Recent interest in gene therapy and manipulation of gene expression hasfocused on the design of synthetic oligonucleotides that can be used toblock or suppress gene expression via an antisense, ribozyme or triplehelix mechanism. To this end, the sequence of the native target DNA orRNA molecule is characterized and standard methods are used tosynthesize oligonucleotides representing the complement of the desiredtarget sequence (see, S. Crooke, The FASEB Journal, Vol. 7, April 1993,p. 533 and references cited therein). Attempts to design more stableforms of such oligonucleotides for use in vivo have typically involvedthe addition of various functional groups, e.g., halogens, azido, nitro,methyl, keto, etc. to various positions of the ribose or deoxyribosesubunits (cf., The Organic Chemistry of Nucleic Acids, Y. Mizuno,Elsevier Science Publishers BV, Amsterdam, The Netherlands, 1987).

2.1.2 Glycopeptides

As a result of recent advances in biological carbohydrate chemistry,carbohydrates increasingly are being viewed as the components of livingsystems with the enormously complex structures required for the encodingof the massive amounts of information needed to orchestrate theprocesses of life, e.g., cellular recognition, immunity, embryonicdevelopment, carcinogenesis and cell-death. Thus, whereas two naturallyoccurring amino acids can be used by nature to convey 2 fundamentalmolecular messages, i.e., via formation of the two possible dipeptidestructures, and four different nucleotides convey 24 molecular messages,two different monosaccharide subunits can give rise to 11 uniquedisaccharides, and four dissimilar monosaccharides can give rise to upto 35,560 unique tetramers each capable of functioning as a fundamentaldiscreet molecular messenger in a given physiological system.

The gangliosides are examples of the versatility and effect with whichorganisms can use saccharide structures. These molecules are glycolipids(sugar-lipid composites) and as such are able to position themselves atstrategic locations on the cell wall: their lipid component enables themto anchor in the hydrophobic interior of the cell wall, positioningtheir hydrophilic component in the aqueous extracellular millieu. Thusthe gangliosides (like many other saccharides) have been chosen to actas cellular sentries: they are involved in both the inactivation ofbacterial toxins and in contact inhibition, the latter being the complexand poorly understood process by which normal cells inhibit the growthof adjacent cells, a property lost in most tumor cells. The structure ofganglioside GM, a potent inhibitor of the toxin secreted by the choleraorganism, featuring a branched complex pentameric structure is shownbelow.

The oligosaccharide components of the glycoproteins (sugar-proteincomposites) responsible for the human blood-group antigens (the A, B,and O blood classes) are shown below.

Interactions involving complementary proteins and glycoproteins on redblood cells belonging to incompatible blood classes cause formation ofaggregates, or clusters and are the cause for failed transfusions ofhuman blood.

Numerous other biological processes and macromolecules are controlled byglycosylation (i.e., the covalent linking with sugars). Thus,deglycosylation of erythropoetin causes loss of the hormone's biologicalactivity; deglycosylation of human gonadotropic hormone increasesreceptor binding but results in almost complete loss of biologicalactivity (see Rademacher et al., Ann. Rev. Biochem 57, 785 (1988); andglycosylation of three sites in tissue plasminogen activating factor(TPA) produces a glycopolypeptide which is 30% more active than thepolypeptide that has been glycosylated at two of the sites.

2.1.3 Design and Synthesis of Mimetics of Biological Ligands

A currently favored strategy for development of agents which can be usedto treat diseases involves the discovery of forms of ligands ofbiological receptors, enzymes, or related macromolecules, which mimicsuch ligands and either boost, i.e., agonize, or suppress, i.e.,antagonize the activity of the ligand. The discovery of such desirableligand forms has traditionally been carried out either by randomscreening of molecules (produced through chemical synthesis or isolatedfrom natural sources), or by using a so-called “rational” approachinvolving identification of a lead-structure, usually the structure ofthe native ligand, and optimization of its properties through numerouscycles of structural redesign and biological testing. Since most usefuldrugs have been discovered not through the “rational” approach butthrough the screening of randomly chosen compounds, a hybrid approach todrug discovery has recently emerged which is based on the use ofcombinatorial chemistry to construct huge libraries of randomly-builtchemical structures which are screened for specific biologicalactivities. (S. Brenner and R. A. Lerner, 1992, Proc. Natl. Acad. Sci.USA 89:5381)

Most lead-structures which have been used in “rational” drug design arenative polypeptide ligands of receptors or enzymes. The majority ofpolypeptide ligands, especially the small ones, are relatively unstablein physiological fluids, due to the tendency of the peptide bond toundergo facile hydrolysis in acidic media or in the presence ofpeptidases. Thus, such ligands are decisively inferior in apharmacokinetic sense to nonpeptidic compounds, and are not favored asdrugs. An additional limitation of small peptides as drugs is their lowaffinity for ligand acceptors. This phenomenon is in sharp contrast tothe affinity demonstrated by large, folded polypeptides, e.g. proteins,for specific acceptors, e.g. receptors or enzymes, which is in thesubnanomolar range. For peptides to become effective drugs, they must betransformed into nonpeptidic organic structures, i.e., peptide mimetics,which bind tightly, preferably in the nanomolar range, and can withstandthe chemical and biochemical rigors of coexistence with biologicalfluids.

Despite numerous incremental advances in the art of peptidomimeticdesign, no general solution to the problem of converting apolypeptide-ligand structure to a peptidomimetic has been defined. Atpresent, “rational” peptidomimetic design is done on an ad hoc basis.Using numerous redesign-synthesis-screening cycles, peptidic ligandsbelonging to a certain biochemical class have been converted by groupsof organic chemists and pharmacologists to specific peptidomimetics;however, in the majority of cases the results in one biochemical area,e.g. peptidase inhibitor design using the enzyme substrate as a leadcannot be transferred for use in another area, e.g. tyrosine-kinaseinhibitor design using the kinase substrate as a lead.

In many cases, the peptidomimetics that result from a peptide structurallead using the “rational” approach comprise unnatural α-amino acids.Many of these mimetics exhibit several of the troublesome features ofnative peptides (which also comprise α-amino acids) and are, thus, notfavored for use as drugs. Recently, fundamental research on the use ofnonpeptidic scaffolds, such as steroidal or sugar structures, to anchorspecific receptor-binding groups in fixed geometric relationships havebeen described (see for example Hirschmann, R. et al., 1992 J. Am. Chem.Soc., 114:9699-9701; Hirschmann, R. et al., 1992 J. Am. Chem. Soc.,114:9217-9218); however, the success of this approach remains to beseen.

In an attempt to accelerate the identification of lead-structures, andalso the identification of useful drug candidates through screening ofrandomly chosen compounds, researchers have developed automated methodsfor the generation of large combinatorial libraries of peptides andcertain types of peptide mimetics, called “peptoids”, which are screenedfor a desirable biological activity. For example, the method of H. M.Geysen, (1984 Proc. Natl. Acad. Sci. USA 81:3998) employs a modificationof Merrifield peptide synthesis wherein the C-terminal amino acidresidues of the peptides to be synthesized are linked to solid-supportparticles shaped as polyethylene pins; these pins are treatedindividually or collectively in sequence to introduce additionalamino-acid residues forming the desired peptides. The peptides are thenscreened for activity without removing them from the pins. Houghton,(1985, Proc. Natl. Acad. Sci. USA 82:5131; and U.S. Pat. No. 4,631,211)utilizes individual polyethylene bags (“tea bags”) containing C-terminalamino acids bound to a solid support. These are mixed and coupled withthe requisite amino acids using solid phase synthesis techniques. Thepeptides produced are then recovered and tested individually. Fodor etal., (1991, Science 251:767) described light-directed, spatiallyaddressable parallel-peptide synthesis on a silicon wafer to generatelarge arrays of addressable peptides that can be directly tested forbinding to biological targets. These workers have also developedrecombinant DNA/genetic engineering methods for expressing huge peptidelibraries on the surface of phages (Cwirla et al., 1990, Proc. Natl.Acad. Sci. USA 87:6378).

In another combinatorial approach, V. D. Huebner and D. V. Santi (U.S.Pat. No. 5,182,366) utilized functionalized polystyrene beads dividedinto portions each of which was acylated with a desired amino acid; thebead portions were mixed together and then split into portions each ofwhich was subjected to acylation with a second desirable amino acidproducing dipeptides, using the techniques of solid phase peptidesynthesis. By using this synthetic scheme, exponentially increasingnumbers of peptides were produced in uniform amounts which were thenseparately screened for a biological activity of interest.

Zuckerman et al., (1992, Int. J. Peptide Protein Res. 91:1) also havedeveloped similar methods for the synthesis of peptide libraries andapplied these methods to the automation of a modular synthetic chemistryfor the production of libraries of N-alkyl glycine peptide derivatives,called “peptoids”, which are screened for activity against a variety ofbiochemical targets. (See also, Symon et al., 1992, Proc. Natl. Acad.Sci. USA 89:9367). Encoded combinatorial chemical syntheses have beendescribed recently (S. Brenner and R. A. Lerner, 1992, Proc. Natl. Acad.Sci. USA 89:5381).

In addition to the lead structure, a very useful source of informationfor the realization of the preferred “rational” drug discovery is thestructure of the biological ligand acceptor which, often in conjunctionwith molecular modelling calculations, is used to simulate modes ofbinding of the ligand with its acceptor; information on the mode ofbinding is useful in optimizing the binding properties of thelead-structure. However, finding the structure of the ligand acceptor,or preferably the structure of a complex of the acceptor with a highaffinity ligand, requires the isolation of the acceptor or complex inthe pure, crystalline state, followed by x-ray crystallographicanalysis. The isolation and purification of biological receptors,enzymes, and the polypeptide substrates thereof are time-consuming,laborious, and expensive; success in this important area of biologicalchemistry depends on the effective utilization of sophisticatedseparation technologies.

Crystallization can be valuable as a separation technique but in themajority of cases, especially in cases involving isolation of abiomolecule from a complex biological milieu, successful separation ischromatographic. Chromatographic separations are the result ofreversible differential binding of the components of a mixture as themixture moves on an active natural, synthetic, or semisynthetic surface;tight-binding components in the moving mixture leave the surface last enmasse resulting in separation.

The development of substrates or supports to be used in separations hasinvolved either the polymerization/crosslinking of monomeric moleculesunder various conditions to produce fabricated materials such as beads,gels, or films, or the chemical modification of various commerciallyavailable fabricated materials e.g., sulfonation of polystyrene beads,to produce the desired new materials. In the majority of cases, priorart support materials have been developed to perform specificseparations or types of separations and are thus of limited utility.Many of these materials are incompatible with biological macromolecules,e.g., reverse-phase silica frequently used to perform high pressureliquid chromatography can denature hydrophobic proteins and otherpolypeptides. Furthermore, many supports are used under conditions whichare not compatible with sensitive biomolecules, such as proteins,enzymes, glycoproteins, etc., which are readily okdenaturable andsensitive to extreme pH's. An additional difficulty with separationscarried out using these supports is that the separation results areoften support-batch dependent, i.e. they are irreproducible.

Recently a variety of coatings and composite-forming materials have beenused to modify commercially available fabricated materials into articleswith improved properties; however the success of this approach remainsto be seen.

If a chromatographic support is equipped with molecules which bindspecifically with a component of a complex mixture, that component willbe separated from the mixture and may be released subsequently bychanging the experimental conditions (e.g. buffers, stringency, etc.)This type of separation is appropriately called affinity chromatographyand remains an extremely effective and widely used separation technique.It is certainly much more selective than traditional chromatographictechniques, e.g. chromatography on silica, alumina, silica or aluminacoated with long-chain hydrocarbons, polysaccharide and other types ofbeads or gels which in order to attain their maximum separatingefficiency need to be used under conditions that are damaging tobiomolecules, e.g. conditions involving high pressure, use of organicsolvents and other denaturing agents, etc.

The development of more powerful separation technologies dependssignificantly on breakthroughs in the field of materials science,specifically in the design and construction of materials that have thepower to recognize specific molecular shapes under experimentalconditions resembling those found in physiological media, i.e. theseexperimental conditions must involve an aqueous medium whose temperatureand pH are close to the physiological levels and which contains none ofthe agents known to damage or denature biomolecules. The construction ofthese “intelligent” materials frequently involves the introduction ofsmall molecules capable of specifically recognizing others into existingmaterials, e.g. surfaces, films, gels, beads, etc., by a wide variety ofchemical modifications; alternatively molecules capable of recognitionare converted to monomers and used to create the “intelligent” materialsthrough polymerization reactions.

3. SUMMARY

A new approach to the construction of novel molecules is described. Thisapproach involves the development of aminimide-based molecular buildingblocks, containing appropriate atoms and functional groups, which may bechiral and which are used in a modular assembly of molecules withtailored properties; each module contributing to the overall propertiesof the assembled molecule. The aminimide building blocks of theinvention can be used to synthesize novel molecules designed to mimicthe three-dimensional structure and function of native ligands, and/orinteract with the binding sites of a native receptor. This logicalapproach to molecular construction is applicable to the synthesis of alltypes of molecules, including but not limited to mimetics of peptides,proteins, oligonucleotides, carbohydrates, lipids, polymers and tofabricated materials useful in materials science. It is analogous to themodular construction of a mechanical device that performs a specificoperation wherein each module performs a specific task contributing tothe overall operation of the device.

The invention is based, in part, on the following insights of thediscoverer. (1) All ligands share a single universal architecturalfeature: they consist of a scaffold structure, made e.g. of amide,carbon-carbon, or phosphodiester bonds which support several functionalgroups in a precise and relatively rigid geometric arrangement. (2)Binding modes between ligands and receptors share a single universalfeature as well: they all involve attractive interactions betweencomplementary structural elements, e.g., charge- and π-typeinteractions, hydrophobic and Van der Waals forces, hydrogen bonds. (3)A continuum of fabricated materials exists spanning a dimensional rangefrom 100 Å to 1 cm in diameter comprising various materials of variedconstruction, geometries, morphologies and functions, all of whichpossess the common feature of a functional surface which is presented toa biologically active molecule or a mixture of molecules to achieverecognition between the molecule (or the desired molecule in a mixture)and the surface. And (4) Aminimide structures, which have remainedrelatively unexplored in the design and synthesis of biologically activecompounds and especially of drugs, would be ideal building blocks forconstructing backbones or scaffolds bearing the appropriate functionalgroups, that either mimic desired ligands and/or interact withappropriate receptor binding sites; furthermore, aminimide modules maybe utilized in a variety of ways across the continuum of fabricatedmaterials described above to produce new materials capable of specificmolecular recognition. These aminimide building blocks may be chirallypure and can be used to synthesize molecules that mimic a number ofbiologically active molecules, including but not limited to peptides,proteins, oligonucleotides, polynucleotides, carbohydrates, lipids, anda variety of polymers and fabricated materials that are useful as newmaterials, including but not limited to solid supports useful in columnchromatography, catalysts, solid phase immunoassays, drug deliveryvehicles, films, and “intelligent” materials designed for use inselective separations of various components of complex mixtures.

Working examples describing the use of aminimide-based modules in themodular assembly of a variety of molecular structures are given. Themolecular structures include functionalized silica surfaces useful inthe optical resolution of racemic mixtures; peptide mimetics whichinhibit human elastase, protein-kinase, and the HIV protease; polymersformed via free-radical or condensation polymerization ofaminimide-containing monomers; and lipid-mimetics useful in thedetection, isolation, and purification of a variety of receptors.

In accordance with the objectives of the present invention, theaminimide-based molecules of interest possess the desiredstereochemistry and, when required, are obtained optically pure. Inaddition to the synthesis of single molecular entities, the synthesis oflibraries of aminimide-based molecules, using the techniques describedherein or modifications thereof which are well known in the art toperform combinatorial chemistry, is also within the scope of theinvention. Furthermore, the aminimide-containing molecules possessenhanced hydrolytic and enzymatic stabilities, and in the case ofbiologically active materials, are transported to target ligand-acceptormacromolecules in vivo without causing any serious side-effects.

4. DETAILED DESCRIPTION OF THE INVENTION

To the extent necessary to further understand any portion of thedetailed description, the following earlier filed U.S. patentapplications are expressly incorporated herein by reference thereto:AMINIMIDE COMPOSITIONS AND BIOLOGICALLY USEFUL DERIVATIVES THEREOF, Ser.No. 07/906,770, filed Jun. 30, 1992; AMINIMIDE-BASED SUPPORT MATERIALSAND FUNCTIONALIZED SURFACES, Ser. No. 07/906,769, filed Jun. 30, 1992;DIRECTED PURE CHIRAL-ISOMER LIGANDS, RECOGNITION AGENTS AND FUNCTIONALLYUSEFUL MATERIALS FROM SUBSTITUTED AMINIMIDES AND DERIVATIVES CONTAININGAN ASYMMETRIC CENTER, Ser. No. 08/041,559, filed Apr. 2, 1993.

4.1 Physical and Chemical Properties of the Aminimide Functional Group

Aminimides are zwitterionic structures described by the resonance hybridof the two energetically comparable Lewis structures shown below.

The tetrasubstituted nitrogen of the aminimide group can be asymetricrendering aminimides chiral as shown by the two enantiomers below.

As a result of the polarity of their structures, but lack of net charge,simple aminimides are freely soluble in both water and (especially)organic solvents.

Dilute aqueous solutions of aminimides are neutral and of very lowconductivity; aminimide conjugate acids are weakly acidic, pK_(a)≅4.5. Astriking property of aminimides is their hydrolytic stability, underacidic, basic, or enzymatic conditions. For example, boiling trimethylamine benzamide in 6 N NaOH for 24 hrs leaves the aminimide unchanged.Upon thermolytic treatment, at temperatures exceeding 180° C.,aminimides decompose to give isocyanates as follows.

4.1.1 Use of the Aminimide Group as a Mimetic of the Amide Group

The aminimide group mimics several key structural features of the amidegroup, such as overall geometry (e.g. both functional groups contain aplanar carbonyl unit and a tetrahedral atom linked to the acylatednitrogen) and aspects of charge distribution (e.g. both functionalgroups contain a carbonyl with significant negative charge developmenton the oxygen). These structural relationships can be seen below, wherethe resonance hybrids of the two groups are drawn.

Being hydrolytically and enzymatically more stable than amides andpossessing novel solubility properties due to their zwitterionicstructures, aminimides are valuable building blocks for the constructionof mimetics of biologically active molecules with superiorpharmacological properties. For the construction of these mimetics, theaminimide backbone is used as a scaffold for the geometrically preciseattachment of structural units possessing desired stereochemical andelectronic features, such as suitable chiral atoms, hydrogen-bondingcenters, hydrophobic and charged groups, π-systems, etc. Furthermore,multiple aminimide units can be linked in a variety of modes, usinglikers of diverse structures, to produce polymers of a great variety ofstructures. Specific molecular forms are chosen for screening andfurther study using several criteria. In one instance a certainaminimide structure is chosen because it is novel and has never beentested for activity as a biopharmaceutical agent or as material fordevice construction. In a preferable instance an aminimide ligand ischosen because it incorporates structural features and propertiessuggested by a certain biochemical mechanism. In another preferable casethe aminimide structure is the result of assembly of molecular moduleseach making a specific desirable contribution to the overall propertiesof the aminimide-containing molecule.

Summarizing, aminimides are functional groups with unusual and verydesirable physiochemical properties, which can be used as molecularmodules for the construction of molecular structures that are useful asbiopharmaceutical agents and as new materials for high technologicalapplications.

4.2 General Synthetic Routes to Aminimides

4.2.1 Aminimides via Alkylation of N,N-Disubstituted Hydrazones

Alkylation of an acyl hydrazide (hydrazone) followed by neutralizationwith a base produces an aminimide.

This alkylation is carried out in a suitable solvent such as ahydroxylic solvent e.g. water, ethanol, isopropanol or a dipolar aproticsolvent e.g., DMF, DMSO, acetonitrile, usually with heating.

The acyl hydrazide is produced by the reaction of a 1,1-disubstitutedhydrazine with an activated acyl derivative or an isocyanate, in asuitable organic solvent, e.g. methylene chloride, toluene, ether, etc.in the presence of a base such as triethylamine to neutralize thehaloacid generated during the acylation.

Activated acyl derivatives include acid chlorides, chlorocarbonates,chlorothiocarbonates, etc.; the acyl derivative may also be replacedwith a suitable carboxylic acid and a condensing agent such asdicyclohexylcarbodiimide (DCC).

The alkylating agent R³X used in the hydrazone alkylation may be analkyl halide (X=Cl, Br, I), a tosylate (X=OTs), or some other suitablereactive species, such as an epoxide. The conversion of phenylisocyanate to an aminimide using the commercially available1,1-dimethylhydrazine and ethylene oxide as the hydrazone alkylatingagent is given below:

The desired 1,1-disubstituted hydrazines may be readily prepared in anumber of ways well known in the art; one is the reaction of a secondaryamine with NH₂Cl in an inert organic solvent.

The above route to aminimides is broadly applicable and allows theincorporation of a wide variety of aliphatic, aromatic and heterocyclicgroups into various positions in the aminimide structure.

4.2.2 Aminimides via Acylation of 1,1,1-Trialkyl Hydrazinium Salts

Acylation of a suitable trialkyl hydrazinium salt by an acyl derivativeor isocyanate in the presence of a strong base in a suitable organicsolvent, e.g. dioxane, ether, acetonitrile, etc. produces good yields ofaminimides.

The acyl derivatives for the acylation reaction are the same as thoserequired for the synthesis of the hydrazones outlined above.

The required hydrazinium salts may be prepared by routine alkylation ofa 1,1-disubstituted hydrazines or by treatment of a tertiary amine witha haloamine (see 78 J. Am. Chem. Soc. 1211 (1956)).

Hydrazinium salts, being chiral at nitrogen, may be resolved, e.g. bytreatment with a chiral acid followed by separation of the diastereomers(e.g. using chromatography or fractional crystallization and theresulting enantiomers used in stereoselective syntheses of aminimides.

When one of the alkyl groups in a hydrazinium salt is an ester group,the ester may be saponified efficiently using LiOH in a mixture ofmethanol and water, producing a useful α-hydrazinium acid afterneutralization of the reaction mixture with an acid.

Suitably protected hydrazinium carboxylates may be used in condensationreactions to produce aminimides. Procedures analogous to those known tobe useful in effecting peptide bond formation are expected to be useful;e.g. DCC or other carbodiimides may be used as condensing agents insolvents such as DMF.

Alternatively, the hydrazinium carboxylate units may be coupled withα-amino-acids or with other nucleophiles, such as amines, thiols,alcohols, etc., using standard techniques, to produce molecules of wideutility as ligand mimetics and new materials for high technologicalapplications.

The α-hydrazinium esters may in turn be produced by the alkylation of a1,1-disubstituted hydrazine with a haloester under standard reactionconditions, such as those given above for the alkylation of hydrazones.

Alternatively, these hydrazinium esters may be produced by standardalkylation of the appropriate α-hydrazino ester.

The required 1,1-disubstituted hydrazine for the above reaction may beobtained by acid or base hydrolysis of the corresponding hydrazone (see108 J. Am. Chem. Soc. 6394 (1986)); the alkylated hydrazone is producedfrom the monosubstituted hydrazone by the method of Hinman and Flores(24 J. Org. Chem. 660 (1958)).

The monosubstituted hydrazones required above may be obtained byreduction of the Schiff base formed from an α-keto ester and a suitablehydrazone. This reduction may also be carried out stereoselectively, ifdesired, using DuPHOS-Rhodium catalysis (114 J. Am. Chem. Soc. 6266(1992); 259 Science 479 (1993)), as shown:

In a variation of the synthesis given above, α-halo aminimides areprepared using an α-halo acyl halide.

(CH₃)₃N⁺NH₂ OTs⁻ClCH₂COCl→(CH₃)₃N⁺N⁻COCH₂Cl

These halo aminimides may be reacted with nucleophiles containingreactive hydroxyl, thio, or amino groups to give complex aminimidestructures.

4.2.3 Aminimides via the Hydrazine-Epoxide-Ester Reaction

A very useful and versatile synthesis of aminimides involves the one-potreaction of an epoxide, an asymetrically disubstituted hydrazine, and anester in a hydroxylic solvent, usually water or an alcohol, which isallowed to proceed usually at room temperature over several hours toseveral days.

In the equation above, R¹, R² and R³ are selected from a set of diversestructural types (e.g. alkyl, cycloalkyl, aryl, aralkyl, alkaryl or manysubstituted versions thereof), and R⁴ and R⁵ are alkyl or cycloalkyl.

The rates for the above reaction increase with increasingelectrophilicity of the ester component. Generally, a mixture of 0.1 molof each of the reactants in 50-100 ml of an appropriate solvent isstirred for the required period at room temperature (the reaction may bemonitored by thin layer chromatography). At the end of this period thesolvent is removed in vacuo to give the crude product.

If substituent R⁴ of the ester component in the above aminimideformation contains a double bond, an aminimide with a terminal doublebond results which may be epoxidized, e.g. using a peracid understandard reaction conditions, and the resulting epoxide used as startingmaterial for a new aminimide formation; thus a structure containing twoaminimide subunits results. If the aminimide-formation and epoxidationsequence is repeated n times, a structure containing n aminimidesubunits results; thus for R⁴=propene, n repetition of the sequenceresults in the structure shown below:

where the designations R² and R³ are used to illustrate the manner inwhich the hydrazine substituents R² and R³ can be varied in eachpolymerization step to produce oligomers or polymers of diversestructures.

A related aminimide polymerization sequence utilizes an ester moietybonded directly to the epoxide group.

An additional related polymerization sequence involves the use ofbifunctional epoxides and esters of the following form

so as to produce polymers of the following structure (shown for the caseof reaction with dimethyl hydrazine):

where X and Y are alkyl, cycloalkyl, aryl, aralkyl or alkaryl linkers.

4.2.4 Synthesis of Enantiomerically-Pure Aminimides

Enantiomerically-pure aminimides may be produced by acylation of chiralhydrazinium salts as shown in the example below.

Chirally-pure hydrazinium salts may be obtained by resolution of theracemates; resolution can be effected by forming salts with opticallypure acids, e.g. tartaric acid, and separating the resultingdiastereomers by means of chromatography or fractional crystallization(see, e.g., 103 J. Chem. Soc. 604 (1913)); alternatively the racemicmodification is resolved by subjecting it to chromatographic separationusing a chiral stationary chromatographic support, or if feasible, bythe use of a suitable enzyme system.

Alternatively, enantiomerically-pure aminimides may be obtained byresolution of the racemic modifications using one of the techniquesdescribed above for the resolution of racemic hydrazinium salts (for anexample, see 28 J. Org. Chem. 2376 (1963)).

An additional approach to the synthesis of chiral aminimides involveschiral synthesis; an example is provided by the reaction of(S)-(−)-propylene oxide with 1,1-dimethylhydrazine andmethyl-(R)-3-hydroxybutyrate, all of which are commercially available.

A variety of chiral epoxides, produced by chiral epoxidations such asthose developed by Sharpless (Asynn. Syn., J. D. Morrison ed., Vol. 5,Ch. 7+8, Acad. Press, New York, N.Y., 1985), and chiral esters, producedby standard procedures, may be used to produce a wide variety of chiralaminimides.

Chirally-pure aminimide molecular building blocks are especiallypreferred since they will be used to produce a vast array of moleculesuseful as new materials for high technological applications and asmolecular recognition agents, including biological ligand mimetics to beused as drugs, diagnostics, and separation agents.

4.4 Synthesis of Specific Classes of Aminimides

4.4.1 Synthesis of Chiral Aminimide-Containing Conjugates

The synthetic routes outlined above may be utilized to produce a widevariety of chiral aminimide conjugates of the following generalstructure:

The substituents A and B shown may be of a variety of structures and maydiffer markedly in their physical or functional properties, or may bethe same; they may also be chiral or symmetric. A and B are preferablyselected from

1) an amino acid derivative of the form (AA)_(n), which would includenatural and synthetic amino acid residues (n=1), peptides (n=2-30),polypeptides (n=31-70) and proteins (n>70).

2) a nucleotide derivative of the form (NUCL)_(n), which would includenatural and synthetic nucleotides (n=1), nucleotide probes (n=2-25) andoligonucleotides (n>25) including both deoxyribose (DNA) and ribose(RNA) variants.

3) a carbohydrate derivative of the form (CH)_(n). This would includenatural physiologically active carbohydrates (glucose, galactose, etc.)including related compounds such as sialic acids, etc. (n=1), syntheticcarbohydrate residues and derivatives of these (n=1) and all of thecomplex oligomeric permutations of these as found in nature (n>1) cf.Scientific American, January 1993, p. 82.

4) a naturally occurring or synthetic organic structural motif. Thisterm includes any of the well known base structures of pharmaceuticalcompounds including pharmacophores or metabolites thereof. Thesestructural motifs are generally known to have specific desirable bindingproperties to ligand acceptors of interest and would include structuresother than those recited above in 1), 2) and 3).

5) a reporter element such as a natural or synthetic dye or a residuecapable of photographic amplification which possesses reactive groupswhich may be synthetically incorporated into the oxazolone structure orreaction scheme and may be attached through the groups without adverselyinterfering with the reporting functionality of the group. Preferredreactive groups are amino, thio, hydroxy, carboxylic acid, acidchloride, isocyanate alkyl halides, aryl halides and oxirane groups.

6) an organic moiety containing a polymerizable group such as a doublebond or other functionalities capable of undergoing condensationpolymerization or copolymerization. Suitable groups include vinylgroups, oxirane groups, carboxylic acids, acid chlorides, esters,amides, lactones and lactams.

7) a macromolecular component, such as a macromolecular surface orstructures which may be attached to the oxazolone modules via thevarious reactive groups outlined above in a manner where the binding ofthe attached species to a ligand-receptor molecule is not adverselyaffected and the interactive activity of the attached functionality isdetermined or limited by the macromolecule. The molecular weight ofthese macromolecules may range from about 1000 Daltons to as high aspossible. They may take the form of nanoparticles (d_(p)=100-1000 Å),latex particles (d_(p)=1000 Å5000 Å), porous or non-porous beads(d_(p)=0.5μ-1000μ), membranes, gels, macroscopic surfaces orfunctionalized or coated versions or composites of these.

Under certain circumstances, A and/or B may be a chemical bond to asuitable organic moiety, a hydrogen atom, an organic moiety whichcontains a suitable electrophilic group, such as an aldehyde, ester,alkyl halide, ketone, nitrile, epoxide or the like, a suitablenucleophilic group, such as a hydroxyl, amino, carboxylate, amide,carbanion, urea or the like, or one of the R groups defined below. Inaddition, A and B may join to form a ring or structure which connects tothe ends of the repeating unit of the compound defined by the precedingformula or may be separately connected to other moieties.

A more generalized presentation of the composition of the invention isdefined by the structure

wherein:

a. at least one of A and B are as defined above and A and B areoptionally connected to each other or to other compounds;

b. X and Y are the same or different and each represents a chemical bondor one or more atoms of carbon, nitrogen, sulfur, oxygen or combinationsthereof;

c. R and R′ are the same or different and each is an alkyl, cycloalkyl,aryl, aralkyl or alkaryl group or a substituted or heterocyclicderivative thereof, wherein R and R′ may be different in adjacent nunits and have a selected stereochemical arrangement about the carbonatom to which they are attached;

d. G is a chemical bond or a connecting group that includes a terminalcarbon atom for attachment to the quaternary nitrogen and G may bedifferent in adjacent n units; and

e. n≧1.

Preferably, if G is a chemical bond, Y includes a terminal carbon atomfor attachment to the quaternary nitrogen; and if n is 1 and X and Y arechemical bonds, R and R′ are the same, A and B are different and one isother than H or R.

In one embodiment of the invention, at least one of A and B represent anorganic or inorganic macromolecular surface. Examples of preferredmacromolecular surfaces include ceramics such as silica and alumina,porous and nonporous beads, polymers such as a latex in the form ofbeads, membranes, gels, macroscopic surfaces or coated versions orcomposites or hybrids thereof. This functionalized surface may berepresented as follows:

In a further embodiment of the invention, the above roles of A and B arereversed, so that B is the substituent selected from the foregoing listand A represents a functionalized surface, as shown below:

In a third preferred embodiment of the invention, either A, B, or bothcontain one or more double bonds capable of undergoing free-radicalpolymerization or copolymerization to produce achiral or chiraloligomers, polymers, copolymers, etc.

Another embodiment of the invention relates to a composition having thestructure

wherein A, Y, R, R¹ and G are as defined above and W is —H

or —H₂X⁻ where X⁻ is an anion, such as a halogen or tosyl anion.

Yet another aspect of the invention relates to a lipid mimeticcomposition having the structure

wherein Q is a chemical bond; an electrophilic group; a nucleophilicgroup; R; an amino acid derivative; a nucleotide derivative; acarbohydrate derivative; an organic structural motif; a reporterelement; an organic moiety containing a polymerizable group; amacromolecular component; or the substituent X(T) or X(T)₂; wherein R isan alkyl, cycloalkyl, aryl, aralkyl or alkaryl group or a substituted orheterocyclic derivative thereof, and T is a linear or branchedhydrocarbon having between 12 and 20 carbon atoms some of which areoptionally substituted with oxygen, nitrogen or sulfur atoms or by anaromatic ring; and provided that at least two T substituents are presentin the structure of the composition.

In the description that follows, R^(n) where n is an integer will beused to designate a group from the definition of R and R¹.

Another aspect of the invention relates to functionalized polymershaving the structure:

wherein

a. X and Y are connecting groups;

b. R^(n) or R′^(n) (where n=an integer) each represent alkyl,cycloalkyl, aryl, aralkyl and alkaryl;

c. (STRUCTURE) is a macromolecular component; and

d. n≧1.

The invention also contemplates various methods of producing anaminimide-functional support. One method comprises the steps of reactinga polymer or oligomer containing pendant moieties of OH, NH or SH with acompound of the formula:

wherein R¹ and R² each represent alkyl, cycloalkyl, aryl, aralkyl oralkaryl, and R³ is an amino acid derivative; a nucleotide derivative; acarbohydrate derivative; an organic structural motif; a reporterelement; an organic moiety containing a polymerizable group; or amacromolecular component;

coating the reacted polymer or oligomer onto a support to form a filmthereon; and heating the coated support to crosslink the film.

Another method comprises the steps of coating a mixture ofmultifunctional esters and multifunctional epoxides onto a support toform a film thereon; and reacting the coated support with1,1′-dialkylhydrazine to crosslink the film.

A third method comprises the steps of coating a mixture of anaminimide-functional vinyl monomer, a difunctional vinyl monomer and avinyl polymerization initiator onto a support to form a film thereon;and heating the coating support to form a crosslinked film.

The aminimide-functionalized support prepared according to the previousmethods are another aspect of the invention.

The ability to derivatize an aminimide scaffold in numerous ways usingthe synthetic techniques outlined above as well as those given below,offers a vast array of structures capable of recognizing specificmolecular entities via establishment of specific types of bindinginteractions. Thus the aminimide shown below is in principle capable ofestablishing the following interactions: π-stacking involving the phenylgroup; hydrogen bonds; acid-base interactions involving the anionicnitrogen; salt bridges involving the quarternary nitrogen; stericinteractions with the bulky isopropyl substituent; and hydrophobicinteractions involving the hydrocarbon chain.

As a further example, possible interactions between a recognition targetand a specific supported aminimide are shown below. Experimentalprocedures for the synthesis of specific chiral aminimides are givenbelow.

4.4.2 Sequential Catenation of Aminimide Subunits Producing Sequences ofVarious Sizes

By choosing aminimide building blocks possessing functional groupscapable of establishing predictable binding interactions with targetmolecules, and using synthetic techniques such as those broadlydescribed above to effect catenation (linking) of the building blocks,it is possible to construct sequences of aminimide subunits mimickingselected native oligomers or polymers, e.g. peptides and polypeptides,which have better stability and pharmacokinetic properties than those ofthe native sequencers. Specific syntheses of multisubunit aminimides areoutlined below.

4.4.2.1 Catenation of Aminimide Subunits via Alkylation/Acylation Cycles

The following steps are involved in this synthesis:

1. Acylation of a chiral hydrazinium salt, prepared as described above,with a molecule capable of functioning both as an acylating and as analkylating agent producing an aminimide; BrCH₂COCl and otherbifunctional species, such as bromoalkyl isocyanates, 2-bromoalkyloxazolones, etc., may be used as acylating agents under the reactionconditions given above.

2. Reaction of the product of the above reaction with an asymmetricallydisubstituted hydrazine to form a diastereomeric mixture of aminimidehydrazinium salts under reaction conditions similar to those describedabove.

3. Isolation of the diastereomers produced in step 2 as described above,e.g. by fractional crystallization or by chromatography using techniquesfamiliar to those skilled in the art.

4. Acylation of the desired diastereomer from step 3 with a bifunctionalacyl derivative similar to those listed in step 1 above producing adimeric type structure.

5. Repetition of steps 2, 3 and 4 the required number of times to buildthe desired aminimide subunit sequence.

6. Capping of the assembled sequence if desired, for example, byreaction with an acylating agent, such as acetyl chloride.

The experimental conditions (e.g. reaction-solvent, temperature andtime, and purification procedures for products) for all of the abovereactions were described above and are also well-known and practiced inthe art. As the molecular weight of the products increases (e.g. in step5 above) solubility and reaction-rate problems may develop if thereactions are run under the conditions that successfully gave productsof much smaller molecular weight. As is well known from the art ofpeptide synthesis, this is probably due to conformational (folding)effects and to aggregation phenomena, and procedures found to work inthe related peptide cases are expected to be very useful in the case ofaminimide catenations. For example, reaction solvents such as DMF, orN-methyl pyrollidone, and chaotropic (aggregate-breaking) agents, suchas urea, are expected to be helpful in alleviating reactivity problemsas the molecular-weight of the product increases.

4.4.2.2 Catenation of Aminimide Subunits via Acylation/Alkylation Cycles

The following steps are involved in this synthesis; experimentalconditions for running the reactions are given above.

1. Alkylation of an asymmetrically disubstituted acyl hydrazide,prepared as outlined above, with a molecule capable of functioning bothas an alkylating and an acylating agent to form a racemic mixture ofaminimides; as before the use of BrCH₂COCl is shown below, but otherbifunctional species, such as bromoalkyl isocyanates, 2-bromoalkyloxazolones, etc. may also be used.

2. Reaction of the racemate from above with an asymmetricallydisubstituted hydrazine to form the hydrazone:

3. Resolution of the racemic modification from the previous step asdescribed above.

4. Alkylation of the product from step 3 with a bifunctional moleculecapable of alkylation and acylation, which may be the same as that usedin step 1 or different, to form a mixture of diastereomeric aminimides.

5. Reaction of the diastereomers from step 4 with a suitableasymmetrically disubstituted hydrazine to form the diastereomerichydrazones, as shown:

6. Separation of the diastereomers as described above.

7. Repetition of steps 4, 5 and 6 to build the desired sequence ofaminimide subunits.

8. Capping of the sequence, if desired, using e.g. methyl bromide toproduce a sequence such as shown below.

4.4.2.3 Catenation of Aminimide Subunits Using Hydrazinolysis of anEster in the Presence of an Epoxide

The following steps are involved in this synthesis; experimentalconditions for running the reactions are given above.

1. Formation of an aminimine from the reaction of an 1,1-asymmetricallydisubstituted hydrazine with an epoxide; the reaction is illustrated fora chiral epoxide below (the chiral epoxide may be obtained by e.g. aSharples epoxidation):

The aminimine is normally not isolated, but used directly for thefollowing reaction.

2. The aminimine is reacted-with an ester-epoxide to give an aminimine;for the mixture of diastereomeric aminimides above and the ester-epoxideshown below, the following is obtained.

3. Separation of the diastereomeric aminimides as described above.

4. Reaction of the desired diastereomeric aminimide with anasymmetrically disubstituted hydrazine to form diastereomericaminimide-aminimines:

5. Repetition of steps 2, 3 and 4 above using the appropriate hydrazinesand epoxy-esters in each step to produce the desired aminimide sequence.

6. “Capping” of the final sequence, if desired, by acylation with asimple ester, such as methyl acetate, to produce the designed aminimideligand shown:

4.4.2.4 Catenation of a-Hydrazinium Esters or Carboxylic Acids

The following steps are involved in this synthesis; experimentalconditions for running the reaction are given above.

1. Treatment of a chirally-pure hydrazinium salt (produced as describedabove) with a strong base, such as NaOMe in an alcohol solvent, to formthe amino anion:

2. Addition of an a-hydrazinium ester (again produced as discussedabove) to an appropriately blocked amino-anion-containing mixture fromstep 1 to form the hydrazinium-aminimide, as shown.

In the equation above, B1 is an appropriate protecting group such as BOC(t-butoxy carbonyl.

3. Removal of B1 followed by repetition of steps 1 and 2 the requirednumber of times to obtain the desired aminimide sequence, followed by a“capping” step, using a simple ester as acylating agent.

Alternatively, the α-hydrazinium carboxylic acids may be obtained bytreatment of the esters with LiOH in MeOH/H₂O at room temperature, asdescribed above, and coupled with each other using condensationreactions promoted by DCC or other agents. Protecting groups used intraditional peptide synthesis are expected to be useful here as well.

4.4.3 Synthesis of Aminimide-Containing Peptides and Proteins

Aminimide subunits may be introduced into any position of a polypeptidevia chemical synthesis, using one of the procedures outlined above,including the techniques for dealing with problematic reactions of highmolecular weight species. The resulting hybrid molecules are expected tohave improved properties over the native molecules; for example, theaminimide group may confer greater hydrolytic and enzymatic stability tothe hybrid molecule over its native counterpart.

As an example of a synthesis of an aminimide-modified peptide, themodification of a peptide attached to a Merrifield solid phase synthesissupport by alkylation with aminimide-containing molecule is shown below.

If moiety B contains a functional group which can be used to linkadditional aminimide and natural or unnatural amino acid subunits, e.g.via acylation reactions, complex hybrid structures may be obtained usingthe experimental procedures outlined above.

4.4.4 Synthesis and Polymerization of Chiral Aminimide-ContainingMonomers

The conversion of many of the aminimide structures described above intomonomer building blocks which can be polymerized to give novelmacromolecules, which are useful in a variety of high technologicalapplications, is contemplated. The following synthetic approaches areexpected to be very useful in the production of new materials.

(a) Free-Radical Polymerization of Vinyl Aminimides

Chiral (as well as achiral) vinylaminimide monomers of the generalstructures shown below may be readily prepared, following the proceduresoutlined above, and used in free-radical polymerizations, according toexperimental procedures well-known in the art, to produce a vast arrayof novel polymeric materials.

Additional monomeric structures useful in preferred free radicalpolymerizations include those shown below; they produce polymeric chainscapable of being crosslinked into more rigid structures. The monomersshown below may be prepared using the synthetic procedures outlinedabove, and the polymerization/crosslinking reactions may be run usingstandard polymerization techniques. See, for example, PracticalMacromolecular Organic Chemistry, Braun, Cherdron and Kern, trans. by K.Ivin, 3ed., Vol Z, Harwood Academic Publishers, New York, N.Y. 1984.

The monomers shown above may be polymerized with other alkenes ordienes, which are either commercially available or readily preparedusing standard synthetic reactions and techniques, to furnish copolymerswith novel structures and molecular recognition characteristics.

(b) Condensation Polymerizations Producing Aminimide-ContainingMacromolecules

Sequential condensations of aminimide-forming molecules may be used toproduce a variety of novel polymers of controlled size. An exampleinvolving dimeric epoxides and esters is given below; processesinvolving trimeric and more complex epoxides and esters are alsocontemplated; and experimental conditions for running thesepolymerizations (including techniques for resolving experimentaldifficulties as product molecular weight increases) have been describedabove.

When the polymerization reaction is carried out with moleculesimmobilized on a support, e.g. silica, a support capable of specificmolecular recognition is produced; an example of such a support is givenbelow:

4.4.5 Lipid Mimetics

Aminimide conjugate structures containing two long-chain alkyl groupscapable of producing bilayer membrane structures are preferredembodiments of the present invention. Many uses of these amphiphilic,surface-active compounds are envisioned. They may be used to isolate andstabilize biologically-active molecules from the cell-wall; they areuseful in the construction of affinity chromatography supports for theisolation and purification of amphiphilic macromolecules, e.g.receptors, enzymes, etc.; and they may serve as effective deliverysystems for the administration of drugs.

The structure of one preferred lipid mimetic is shown below.Substituents R may be chosen from a variety of structures of varioussizes including structures of ligands of biological receptors orenzymes; a preferred combination of substituents involves stericallysmall groups for R₁ and R₂ and a group such as A or B described abovefor R₃; the long-chain alkyl groups are 4-20 carbons in length; group Xis a linker composed of atoms chosen from the set of C, H, N, O, and S.

A further desirable variation of the surface-active structure shownabove is as follows:

In the above structure, X is a linker group (e.g. CH); one or moresubstituents R are chosen from the group of structures A and B describedabove and the remaining substituent(s) in preferably a sterically smallgroup, e.g. H, or CH₃. An additional desirable amphiphilic structure isshown below; substituent structures are similar to those listed above.

An example of a synthesis of a lipid mimetic is given below; therequired experimental conditions for the reactions that follow aresimilar to those described above for related transformations.

Thus,

is combined with (CH₃)₂NNH₂ and CH₃COOCH₃ to produceCH₃(CH₂)₁₄CH₂CH(OH)CH₂N(CH₃)₂NCOCH₃, which is then combined withCH₃(CH₂)₁₄CH₂Br under alklaline conditions to produce:

4.4.6 Fabrication of Aminimide-Containing Macromolecular StructuresCapable of Specific Molecular Recognition

In an embodiment of the invention aminimide molecular building blocksmay be utilized to construct new macromolecular structures capable ofrecognizing specific molecules (“intelligent macromolecules'”). The“intelligent macromolecules” may be represented by the following generalformula:

P—C—L—R

where,

R is a structure capable of molecular recognition;

L is a linker;

P is a macromolecular structure serving as a supporting platform;

C is a polymeric structure serving as a coating which surrounds P.

Structure R may be a native ligand or a biological ligand-acceptor or amimetic thereof, such as those described above.

Linker L may be a chemical bond or one of the linker structures listedabove, or a sequence of subunits such as amino acids, aminimidemonomers, oxazolone-derived chains of atoms, etc.

Polymeric coating C may be attached to the supporting platform eithervia covalent bonds or “shrink wrapping,” i.e. the bonding that resultswhen a surface is subjected to coating polymerization well known tothose skilled in the art. This coating element may be

1) a thin crosslinked polymeric film 10-50 Å in thickness;

2) a crosslinked polymeric layer having controlled microporosity andvariable thickness, or

3) a controlled microporosity gel. When the support platform is amicroporous particle or a membrane, as described below, the controlledmicroporosity gel may be engineered to completely fill the porousstructure of the support platform. The polymeric coatings may beconstructed in a controlled way by carefully controlling a variety ofreaction parameters such as the nature and degree of coatingcrosslinking, polymerization initiator, solvent, concentration ofreactants, and other reaction conditions, such as temperature,agitation, etc., in a manner that is well known to those skilled in theart.

The support platform P may be a pellicular material having a diameter(dp) from 100 Å to 1000 μ, a latex particle (dp 0.1-0.2μ), a microporousbead (dp 1-1000μ), a porous membrane, a gel, a fiber, or a continuousmacroscopic surface. These may be commercially available polymericmaterials, such as silica, polystyrene, polyacrylates, polysulfones,agarose, cellulose, etc. or synthetic aminimide-containing polymers suchas those described below.

Any of the elements P, C, L, or R containing an aminimide-basedstructure is derived from a form of the element containing a precursorto the aminimide-based structure. The multisubunit recognition agentsabove are expected to be very useful in the development of targetedtherapeutics, drug delivery systems, adjuvants, diagnostics, chiralselectors, separation systems, and tailored catalysts.

In the present specification the terms “surface”, “substrate”, and“structure” refer to either P, P linked to C or P linked to C and L asdefined above.

Thus, another aspect of the invention relates to a three-dimensionalcrosslinked random copolymer containing, in copolymerized form about 1to 99 parts of a free-radically polymerizable monomer containing anaminimide group; up to 98 parts of a free-radicallyaddition-polymerizable comonomer; and about 1 to 50 parts of at leastone crosslinking monomer.

The comonomer used in this copolymer may be water-soluble orwater-insoluble, and the copolymer is fashioned into a water-insolublebead, a water-insoluble membrane or a latex particle, or can be aswollen aqueous gel suitable for use as an electrophoresis gel.

This copolymer is preferably the reaction product of about 1 to 99 partsof a condensation-polymerizable monomer containing a moiety clusterselected from the group consisting of (1) at least three epoxy groups,(2) at least three ester groups, (3) at least one epoxy and at least twoester groups and (4) at least one ester and at least two epoxy groups;about 1 to 99 parts of a second condensation-polymerizable monomercontaining a moiety cluster selected from the group consisting of (1) atleast two ester groups, (2) at least two epoxy groups and (3) at leastone ester and one epoxy group; and an amount of 1,1-dialkylhydrazineequivalent, on a molar basis, substantially equal to the total molarcontent of epoxy groups.

4.4.6.1 Aminimide Containing Support Materials

Commercially available or readily obtainable chromatographic supportmaterials for chromatographic and other applications, as well as otherfabricated materials may be derivatized with tailored aminimidemoieties, through chemical modification, producing novel materialscapable of recognizing specific molecular structures.

The following general structures are contemplated.

In the structures above, A is selected from the group consisting ofamino acids, oligopeptides, polypeptides and proteins, nucleotides,oligonucleotides, polynucleotides, carbohydrates, molecular structuresassociated with therapeutic agents, metabolites, dyes, photographicallyactive chemicals, and organic structures having desired steric, charge,hydrogen-bonding or hydrophobicity elements; X and Y are chemical bondsor groups consisting of atoms selected from the set of C, H, N, O, S; R¹and R² are chosen from the group of alkyl, cycloalkyl, aryl, aralkyl,alkaryl and, preferably, structures mimicking the side-chains ofnaturally-occurring amino acids.

Surfaces and other structures functionalized with multiple aminimidesubunits are also preferred; general structures are shown below.

In the above structures R^(1 . . . n) and R′^(1 . . . n) are used toillustrate the manner in which the hydrazine substituents R¹ and R² canbe varied in each polymerization step described above to produce afunctional supported oligomer or polymer.

The following chemical modifications can be used to prepareaminimide-functionalized surfaces.

4.4.6.1.1 Functionalization of Ester and Epoxy Surfaces

A surface bearing ester groups can be treated with an epoxide,containing desired group B, and a disubstituted hydrazine to form anaminimide surface as follows:

To carry out the above reaction, the surface is treated with a solutioncontaining a 10% molar excess of the epoxide (based on the calculatednumber of reactive ester groups of the surface), and a stoichiometricamount of the hydrazine (with respect to the amount of the epoxide) inan appropriate solvent, such as an alcohol, with shaking. The mixture isthen allowed to stand at room temperature for 1 week with occasionalshaking. At the end of this period, the solvent is removed bydecantation, and the surface is thoroughly washed with fresh solvent andair dried.

This approach allows the functionalization of readily available supportscontaining ester groups.

The above reaction sequence can also be employed with anepoxide-functionalized surface:

To carry out the above reaction, the surface is treated with a solutioncontaining a 10% molar excess of the ester (based on the calculatednumber of reactive epoxide groups of the support), and a stoichiometricamount of the hydrazine (with respect to the amount of the ester used),in an appropriate solvent, such as an alcohol, with shaking. The mixtureis then allowed to stand at room temperature for 1 week with occasionalshaking. At the end of this period, the solvent is removed bydecantation, and the surface is thoroughly washed with fresh solvent andair dried.

The foregoing reaction can be modified by utilizing an ester whosesubstituent B contains a double bond. After completion of the reactionshown above, the double bond of the ester can be epoxidized using one ofa variety of reactions including the asymetric epoxidation of Sharples(e.g., utilizing a peracid under suitable reaction conditions well-knownin the art), and the product used as the epoxide in a new repetition ofthe aminimide-forming reaction. The overall process can be repeated toform oligomers and polymers.

For example, using β,δ-butenoic acid methyl ester as the ester, nrepetitions of the above reaction sequence produces a compound of theform:

where the designations R^(1 . . . n) and R′^(1 . . . n) are used toillustrate the manner in which the hydrazine substituents R² and R³ arevaried in each polymerization step, if desired, to produce an oligomeror polymer.

The foregoing reactions can be carried out using bifunctional esters ofthe form ROOC—X—COOR′, where X is a linker and R and R′ are alkyl groupsas defined above, and/or bifunctional epoxides of the form shown below,

wherein Y is a linker as defined above to form desirable polymers. If anester-functionalized surface is reacted with bifunctional esters andepoxides, the resulting surface will have the following generalstructure.

If an epoxide-functionalized surface is reacted as above the derivatizedsurface will have the following general structure.

4.4.6.1.2 Functionalization of Amine Surfaces

An amine-functionalized surface can be converted to an ester-bearingsurface by reaction with an acrylic ester as shown in sequence (a)below. This reaction is followed by reaction with hydrazine and anepoxide as shown in sequence (b).

For reaction (a), a 10% molar excess of methyl acrylate (based on thenumber of reactive amino groups the surface as determined by a titrationwith acid) is dissolved in an appropriate solvent, such as an alcohol,and added to the surface. After addition is complete, the mixture isshaken at room temperature for 2 days. The solvent is then removed bydecantation and the surface is washed thoroughly with fresh solvent inpreparation for the next step.

For reaction (b) the stoichiometric amount of a 1:1 mixture of thehydrazine and the epoxide, is combined in an appropriate solvent, suchas an alcohol, and quickly added to the solvent-wet surface fromreaction (a). The mixture is shaken at room temperature for 3 days. Thesolvent is then removed by decantation, and the surface is-washedthoroughly with fresh solvent and dried.

The above reaction sequence can also be employed with anepoxide-functionalized surface, in which case substituent B in thestructure above represents the surface and the desired functional groupbears the amine moiety. One way of obtaining such a surface is to reacta silica surface with a silicic ester containing an epoxide group toproduce a so-called “epoxy silica”, as shown below.

4.4.6.1.3 Functionalization of Carboxylic-Acid-Containing Surfaces

A surface functionalized with a carboxylic acid group can be reactedwith an 1,1-dialkylhydrazine and a coupling agent, such as dicyclohexylcarbodiimide (DCC), to form a hydrazone-containing surface as shown instep (a) below. This surface can then be coupled with a desired group Bbearing a substituent capable of alkylating the hydrazone to give anaminimide structure (after treatment with base), as shown in step (b):

Substituent B is a surface functionalized with an alkylating agentcapable of reacting with a hydrazone.

To perform the above chemical modification of a carboxyl-bearingsurface, the surface is treated with a 10% molar excess equimolaramounts of the N,N-dimethylhydrazine and DCC in a suitable solvent, suchas methylene chloride, and the mixture is shaken for 2 hours at roomtemperature. The slurry is then removed by decantation and the surfaceis washed thoroughly with fresh solvent to remove any residualprecipitated dicyclohexyl urea. The surface is then treated with astoichiometric amount of the alkylating agent in a suitable solvent,warmed to 70° C. and held at this temperature for 6 hours. The mixtureis then cooled, the solvent is removed by decantation, and the surfaceis washed with fresh solvent and dried.

4.4.6.1.4 Functionalization of Surfaces Capable of Hydrazide Alkylation

A surface bearing a group capable of alkylating acyl hydrazones can befunctionalized to contain aminimide groups as follows:

In the equation above, Z and W are linkers composed of atoms selectedfrom the set of C, N, H, O, S, and X is a suitable leaving group, suchas a halogen or tosylate.

A hydrazone bearing a desired group B is produced by reacting theappropriate 1,1′-dialkylhydrazine with any of a variety of derivativescontaining B via reactions that are well-known in the art. Thesederivatives may be acid halides, azlactones (oxazolones), isocyanates,chloroformates, or chlorothioformates.

4.4.6.1.5 Functionalization of Surface Bearing —NH, —SH, or —OH Groupswith Chloromethyl Aminimides

Surfaces functionalized with —NH₂, —SH, or —OH groups can befunctionalized by treating them with chloromethyl aminimides in thepresence of strong base using the experimental conditions outlinedabove:

The required chloromethyl aminimides can be prepared by known literatureprocedures (See, e.g., 21 J. Polymer Sci., Polymer Chem. Ed. 1159(1983)), or by using the techniques described above.

4.4.6.1.6 Functionalization of oxazolone-Containing Surfaces

Oxazolone-containing surfaces can be functionalized by first reactingthem with 1,1′-dialkylhydrazine as shown in step (a) below followed byalkylation of the resulting hydrazone with an alkylating agent B—CH₂—Xas shown in step (b); reaction conditions similar to those describedabove are expected to be effective in carrying out these modifications.

In the structures above, R³ and R⁴ are derived from the five memberedazlactone ring denoted by Az.

Although the previous discussions are specifically directed to thefunctionalization of surfaces, these reactions can also be used toconstruct aminimide linkages to the other species of A and B which aredescribed in this application.

4.4.7 Preparation of Aminimide-Based Coatings for Support Materials

It is possible to produce aminimide-functionalized composite supportmaterials by coating various soluble aminimide formulations on thesurfaces of existing supports, and subsequently crosslinking theresulting coatings in place to form mechanically stable surfaces. Thecoating may be engineered for a particular application (e.g., to takethe form of a thin non-porous film or to possess localized microporosityfor enhanced surface area) by judicious selection of process conditions,monomer loading levels, the crosslinking mechanism and the amount ofcrosslinker.

For example, any of the foregoing reactions can be carried out with avinyl aminimide in contact with a selected surface, which is polymerizedaccording to well-known techniques (see, e.g., U.S. Pat. No. 4,737,560).The polymerization results in a surface coated with a polymer containingaminimide side-chains. Other coating procedures employing aminimidefunctional groups are described below in greater detail.

4.4.8 Synthesis of Aminimide-Containing Materials Via Polymerizations ofAminimide-Based Molecules

In addition to utilizing aminimide chemistry to chemically modifycommercially available or readily obtainable surfaces, new surfaces andother materials can be fabricated de novo from aminimide precursorsbearing polymerizable groups by polymerizations and/or copolymerizationsin the presence or absence of crosslinking agents. Depending upon theproperties for the desired material, various combinations of monomers,crosslinkers, and ratios thereof may be employed. The resultant supportmaterials may be latex particles, porous or non-porous beads, membranes,fibers, gels, electrophoresis gels, or hybrids thereof. Furthermore, themonomers and crosslinking agents may or may not all be aminimides.

Vinyl or condensation polymerizations may be advantageously employed toprepare the desired aminimide-containing materials. Vinyl polymerizationcan include use of one or more monomers of the form CH₂═CH—X that arecopolymerizable with aminimides; suitable examples include styrene,vinyl acetate, and acrylic monomers. If desired, compatiblenon-aminimide crosslinkers, such as divinyl benzene, may be employed(either singly or in combination as the other such agents).

Condensation polymerization may be accomplished using multifunctionalepoxides and multifunctional esters with the appropriate amounts of an1,1′-dialkylhydrazine, using the reaction conditions described above.Either the ester component or the epoxide component should be at leasttrifunctional to obtain three-dimensionally crosslinked polymerstructures; preferably, both components are trifunctional.

The nature and conditions of processing, the ratio of the variousmonomers and the ratio of crosslinker to total monomer content can bevaried to produce a variety of product structures (e.g., beads, fibers,membranes, gels, or hybrids of the foregoing) and to tailor themechanical and surface properties of the final product (e.g., particlesize and shape, porosity, and surface area). Appropriate parameters fora particular application are readily selected by those skilled in theart.

4.4.9 Combinatorial Libraries of Peptidomimetics Derived From AminimideModules

The synthetic transformations of aminimides outlined above may bereadily carried out on solid supports in a manner analogous toperforming solid phase peptide synthesis, as described by Merrifield andothers (see for example, Barany, G., Merrifield, R. B., Solid PhasePeptide Synthesis, in The Peptides Vol. 2, Gross E., Meienhofer, J.eds., p. 1-284, Acad. Press, New York 1980; Stewart, J. M., Yang, J. D.,Solid Phase Peptide Synthesis, 2nd ed., Pierce Chemical Co., Rockford,Ill. 1984; Atherton, E., Sheppard, R. C., Solid Phase Peptide Synthesis,D. Rickwood & B. D. Hames eds., IRL Press ed. Oxford U. Press, 1989).Since the assembly of the aminimide-derived structures is modular, i.e.,the result of serial combination of molecular subunits, hugecombinatorial libraries of aminimide-based oligomeric structures may bereadily prepared using suitable solid-phase chemical synthesistechniques, such as those of described by Lam (K. S. Lam, et al. Nature354, 82 (1991)) and Zuckermann (R. N. Zuckermann, et al. Proc. Natl.Acad. Ser. USA, 89, 4505 (1992); J. M. Kerr, et al., J. Am Chem. Soc.115, 2529 (1993)). Screening of these libraries of compounds forinteresting biological activities, e.g., binding with a receptor orinteracting with enzymes, may be carried out using a variety ofapproaches well known in the art. With “solid phase” libraries (i.e.,libraries in which the ligand-candidates remain attached to the solidsupport particles used for their synthesis) the bead-staining techniqueof Lam may be used. The technique involves tagging the ligand-candidateacceptor (e.g., an enzyme or cellular receptor of interest) with anenzyme (e.g., alkaline phosphatase) whose activity can give rise tocolor production thus staining library support particles which containactive ligand-candidates and leaving support particles containinginactive ligand-candidates colorless. Stained support particles arephysically removed from the library (e.g., using tiny forceps that arecoupled to a micromanipulator with the aid of a microscope) and used tostructurally identify the biologically active ligand in the libraryafter removal of the ligand acceptor from the complex by e.g., washingwith 8M guanidine hydrochloride. With “solution-phase” libraries, theaffinity selection techniques described by Zuckermann above may beemployed.

An especially preferred type of combinatorial library is the encodedcombinatorial library, which involves the synthesis of a unique chemicalcode (e.g., an oligonucleotide or peptide), that is readily decipherable(e.g., by sequencing using traditional analytical methods), in parallelwith the synthesis of the ligand-candidates of the library. Thestructure of the code is fully descriptive of the structure of theligand and used to structurally characterize biologically active ligandswhose structures are difficult or impossible to elucidate usingtraditional analytical methods. Coding schemes for construction ofcombinatorial libraries have been described recently (for example, seeS. Brenner and R. A. Lerner, Proc. Natl. Acad. Sci. USA 89, 5381 (1992);J. M. Kerr, et al. J. Am. Chem. Soc. 115, 2529 (1993)). These and otherrelated schemes are contemplated for use in constructing encodedcombinatorial libraries of oligomers and other complex structuresderived from aminimide units.

The power of combinatorial chemistry in generating screenable librariesof chemical compounds e.g., in connection with drug discovery, has beendescribed in several publications, including those mentioned above. Forexample, using the “split solid phase synthesis” approach outlined byLam et al., the random incorporation of 20 different aminimide unitsinto pentameric structures, wherein each of the five subunits in thepentamer is derived from one of the aminimide units, produces a libraryof 20⁵=3,200,000 peptidomimetic ligand-candidates, each ligand-candidateis attached to one or more solid-phase synthesis support particles andeach such particle contains a single ligand-candidate type. This librarycan be constructed and screened for biological activity in just a fewdays. Such is the power of combinatorial chemistry using oxazolonemodules to construct new molecular candidates.

The following is one of the many methods that are being contemplated foruse in constructing random combinatorial libraries of aminimides-basedcompounds; the random incorporation of three aminimides derived fromα-chloroacetyl chloride and the hydrazines shown below to produce 27trimeric structures linked to the support via a succinoyl linker isgiven below.

(1) A suitable solid phase synthesis support, e.g., the chloromethylresin of Merrifield is treated with 4-hydroxyl butyric acid in thepresence of CsCO₃ followed by tosylation with p-toluenesulfonylchloride, under conditions known in the art;

2) The resulting resin is split into three equal portions. Each portionis coupled with one of the hydrazines shown above to give thehydrazinium resin which is converted to the aminimide by reaction withchloroacetyl chloride using the experimental conditions described above.

(3) The aminimide resin portions are mixed thoroughly and split againinto three equal portions. Each resin portion is coupled with adifferent hydrazine followed by a coupling with α-chloracetyl chlorideproducing a resin with two linked aminimide subunits. The resin portionsare then mixed thoroughly and split into three equal portions.

(4) Each resin portion is coupled with a different hydrazine followed byreaction with an acid chloride to produce a resin with three linkedaminimide subunits;

The resin portions are mixed producing a library containing 27 types ofbeads each bead type containing a single trimeric aminimide species forscreening using the bead-stain method described above. Alternatively,the aminimides may be detached from the support via acidolysis producinga “solution-phase” library of aminimides containing a butyrylatedterminal nitrogen. (Shown in the structure below in which R═C₃H₇)

4.4.10 Design and Synthesis of Aminimide-Based Glycopeptide Mimetics

A great variety of saccharide and polysaccharide structural motifsincorporating aminimide structures are contemplated including, but notlimited to, the following.

(1) Replacement of certain glycosidic linkages by aminimide backbonesusing reactions well known in the art of sugar chemistry and reactionsdescribed above.

(2) Use of aminimide structures as linkers holding in place a sugarderivative and a tailored mimetic, or another sugar.

4.4.11 Design and Synthesis of Aminimide-Containing OligonucleotideMimetics

The art of nucleotide and oligonucleotide synthesis has provided a greatvariety of suitably blocked and activated furanoses and otherintermediates which are expected to be very useful in the constructionof aminimide-based mimetics. (Comprehensive Organic Chemistry, Sir DerekBarton, Chairman of Editorial Board, Vol. 5, E. Haslam, Editor, pp.23-176).

A great variety of nucleotide and oligonucleotide structural motifsincorporating aminimide-based structures are contemplated including, butnot limited to, the following.

(1) For the synthesis of oligonucleotides containing peptidicaminimide-based linkers in place of the phosphate diester groupingsfound in native oligonucleotides, the following approach is one of manythat is expected to be useful.

(2) For the synthesis of structures in which an aminimide grouping isused to link complex oligonucleotide-derived units, an approach such asthe following is expected to be very useful.

5. EXAMPLE: SYNTHESIS OF AN AMPHIPHILIC LIGAND Mimetic Useful inIsolation and Purification of Receptors Binding Vincamine

To a solution of 1.84 g (0.01 mol) of 1,2-epoxy dodecane (I) in asuitable solvent, such as n-propanol, is added with stirring 0.61 g(0.01 mol) of 1,1-dimethylhydrazine. The solution is stirred for 1 hourat room temperature, cooled to 10° C. in an ice bath, and a solution of3.54 g (0.01 mol) of vincamine (II) dissolved in the minimum amount ofthe same solvent is added. The reaction mixture is stirred at 0° C. for2 hours, allowed to come to room temperature, and stirred at roomtemperature for 3 days. At the end of this time the solvent is removedunder high vacuum (0.2 torr) and the crude product is isolated. Theconjugate (II) is useful as a stabilization agent for the isolation andpurification of receptor proteins which are therapeutically acted uponby vincamine and by structurally related molecules.

6. EXAMPLE: Synthesis of Amphiphilic Ligand Mimetic Useful in theIsolation and Purification of Serotonin Binding Receptor

8.61 g (0.1 mol) of methyl acrylate is added over a 15 minute period toa stirred solution of 17.62 g (0.1 mol) of serotonin in 100 ml of asuitable solvent. The reaction mixture is allowed to come to roomtemperature and stirred at room temperature for 2 days. The solvent isthen removed by freeze drying to yield the ester (IV). 6.01 g (0.1 mol)of 1,1-dimethylhydrazine is added with stirring to a solution of 18.4 g(0.1 mol) of 1,2-epoxydodecane in a suitable solvent, such as propanol.The mixture is stirred at room temperature for 1 hour and a solution of(IV) dissolved in the same solvent is added. The mixture is then stirredat room temperature for 3 days. At the end of this time the solvent isremoved in vacuo to yield the serotonin conjugate (V), which is usefulas a ligand for the discovery, stabilization and isolation ofserotonin-binding membrane receptor proteins.

7. EXAMPLE: Synthesis of Rhodamine-B-Containing Ligand Mimetic Useful inthe Isolation and Purification of Codeine-Binding Proteins

49.74 g (0.1 mol) of the acid chloride of Rhodamine B (VI), preparedfrom rhodamine B by the standard techniques for preparing acid chloridesfrom carboxylic acids, are dissolved in 500 ml of a suitable solvent andare added, with stirring, over a 1-hour period to a solution of 6.01 g(0.1 mol) of 1,1-dimethylhydrazine in 100 ml of the same solvent. Thetemperature is kept at 10° C. After the addition is complete, themixture is stirred at room temperature for 12 hours, and the solvent isstripped away in vacuo to yield the Rhodamine B dimethylhydrazine (VII).

5.21 g (0.01 mol) of (VII) is dissolved in 100 ml of a suitable solvent,such as benzene, and 4.69 g (0.01 mol) of tosyl codeine (VIII), preparedfrom codeine by the standard techniques for the tosylation of analcohol, in 50 ml of the same solvent is added over a 15-minute periodwith stirring. The mixture is heated to reflux and held at reflux for 1hour. The mixture is then cooled, the solvent is removed in vacuo, theresidue is redissolved in an appropriate alcohol and adjusted to pH 8with 10% methanolic KOH. The precipitated salts are removed byfiltration and the solvent is stripped in vacuo to yield the conjugate(IX), useful as a probe for the location, stabilization and isolation ofreceptor proteins that bind codeine and structurally similar analogues.

8. EXAMPLE: Synthesis of Disperse-Blue-3-Containing Ligand MimeticUseful in the Isolation and Purification of Codeine-Binding Proteins

To a solution of 0.285 g (0.001 mol) of norcodeine (X) dissolved in 50ml of a suitable solvent (such as benzene) is added a solution of 0.139g (0.001 mol) of 4,4′-dimethylvinylazlactone (XI) in 10 ml of the samesolvent and the resulting solution is heated to 70° C. and held at thistemperature for 10 hrs. At the end of this time the temperature isbrought to 10° C. with cooling and 0.06 g (0.001 mol) of1,1-dimethylhydrazine dissolved in 10 ml of the same solvent is addeddropwise. The solution is then re-heated to 70° C. and held at thistemperature for 2 hours. 0.466 g (0.001 mol) of the Disperse blue 3tosylate (XII), prepared by the standard tosylation techniques from apure sample of the dye (obtained from the commercial material bystandard normal-phase silica chromatography), is added and the mixtureis heated at 70° C. for 2 more hours. The solvent is then removed invacuo, the residue is redissolved in an appropriate alcohol solvent andtitrated to pH 8 (measured with moist pH paper) with 10% (w/v)methanolic KOH. The precipitated salts are then removed by filtration,and the filtrate is stripped in vacuo to give conjugate (XIII), usefulas a probe for the location and isolation of receptor proteins that bindcodeine and similar molecules.

9. EXAMPLE: Synthesis of an Amphiphilic Ligand Mimetic for the Isolationand Purification of Codeine-Binding Proteins

29.95 g (0.1 mol) of octadecylisocyanate is added slowly to 6.01 g (0.1mol) of 1,1-dimethylhydrazine in 100 ml of benzene. The mixture isstirred for 18 hours at room temperature and 54.2 g (0.1 mol) of tosylcodeine (VIII), prepared by the standard techniques, is addedportionwise over a ½-hour period. The mixture is heated to reflux andstirred at reflux for 2 hours. At the end of this time the solvent isremoved in vacuo, the residue is dissolved in an appropriate solvent(such as ethanol), and the pH is titrated to 8 (measured with moist pHpaper) with 10% (w/v) methanolic KOH. The precipitated salts are thenremoved by filtration and the solvent is removed in vacuo to give thecrude conjugate (XIV), useful for stabilizing and isolating receptorproteins that bind to codeine and to similar molecules.

10. EXAMPLE: Synthesis of a Mimetic of the Protein-Kinase BindingPeptide

a. The dodecamer peptide(BEAD)—Asp—His—Ile—Ala—Asn—Arg—Arg—Gly—Thr—Arg—Gly—Ser—NH₂ is obtainedattached to the solid support as shown using standard FMOC peptidesynthesis techniques, after deprotection of the terminal FMOC group.This peptide is shaken with a solution of an equivalent molar amount ofClCH₂COCl in a suitable solvent at 50° C. for 6 hours. The solvent isremoved by decantation, leaving a terminal —NH—CO—CH₂Cl group attachedto the peptide.

b. A solution of equimolar amounts of 1,1-dimethylhydrazine anddicyclohexylcarbodiimide in a suitable solvent is treated with anequivalent molar amount of the heptamer peptideH₂N—Thr—Thr—Tyr—Ala—Asp—Phe—Ile—COOH, prepared and obtained in the freestate using the standard FMOC solid phase peptide synthesis chemistry(e.g., using instruments and methods marketed by the Milligen Divisionof Millipore Corp.). The mixture is stirred for 4 hours at roomtemperature. The precipitated N,N′-dicyclohexyl urea is removed bycentrifuging and decantation, and the solution is added to thefunctionalized beads prepared in a above. The mixture is then heated to50° C. and shaken overnight at this temperature. The mixture is cooled,the solvent removed by decantation, and the peptide released from the,bead to yield the aminimide mimeticH₂N—Thr—Thr—Tyr—Ala—Asp—Phe—Ile—CO—N—N(CH₃)₂—CH₂—Ser—Gly—Arg—Thr—Gly—Arg—Asn—Ala—Ile—His—Asp—COOH.This mimetic has the aminimide in place of alanine in the naturallyoccurring protein-kinase binding peptide, UK (5-24), and is useful as asynthetic binding peptide with enhanced proteolytic stability.

11.1. Synthesis of a Mimetic of a Human Elastase Inhibitor

This example teaches the synthesis of a competitive inhibitor for humanelastase based on the structure of known N-trifluoroacetyl dipeptideanalide inhibitors (see 162 J. Mol. Biol. 645 (1982) and referencescited therein).

To 3.7 g (0.01 mol) of the aminimideN-(p-isopropylanalido)-methyl)-S-N-methyl-N-benzylchloromethylacetamidein 50 ml ethanol was added 1.86 g (0.01 mol) of1-methyl-1-isobutyl-2-N-trifluoroacetyl hydrazone (prepared from thereaction of trifluoroacetic anhydride with 1-methyl-1-isobutylhydrazine[from methylisobutylamine and chloramine] using standard acylationmethods) in 50 ml ethanol. The mixture was heated to reflux and stirredat reflux for 4 hours. The mixture was then cooled to room temperatureand titrated with 10% (w/v) KOH in methanol to the phenolphthaleinendpoint. The mixture was then filtered and the solvent removed in vacuoon a rotary evaporator. The residue was taken up in benzene andfiltered. Removal of the benzene on the rotary evaporator yielded 5.1 g(95%) of crude mixed diastereomeric aminimides. The desired (S)−(S)isomer was obtained by normal-phase chromatographic purification oversilica. This product is useful as a competitive inhibitor for humanelastase, characterized by HPLC on Crownpack™ CR(+) chiral stationaryphase (Daicell Chemical Industries Ltd.) using pH 2 aqueous mobilephase. NMR (DMSO-d₆): Chemical shifts, peak integrations and D₂Oexchange experiments diagnostic for structure.

11.2. Synthesis of the Chiral Chloroaminimide Starting Material

A mixture of 4.2 g (0.01 mol) of the hydrazinium iodide enantiomerprepared as outlined below, 1.0 g (0.0106 mol) chloroacetic acid and1.24 g (0.011 mol) chloracetyl chloride, contained in a micro reactionflask equipped with a drying tube, was heated in an oil bath at 105° C.for 1 hour. The (homogeneous) reaction mixture was then cooled to roomtemperature and extracted with 4×20 ml of ethyl ether to removechloracetyl chloride and chloracetic acid, with vigorous stirring eachtime. The residual semisolid was dissolved in the minimum amount ofmethanol and titrated with 10% KOH in methanol to the phenolphthaleinend point. The precipitated salts were filtered and the filtrateevaporated to dryness on a rotary evaporator at 40° C. The residue wastaken up in benzene and filtered. The solvent was removed on a rotaryevaporator to yield 3.37 g (90%) of the (S)-aminimide enantiomer,characterized by CDCL₃ NMR spectrum and D₂O exchange experiments anddirectly used in the next step in the sequence (see above).

11.3. Synthesis of the Chiral Aminimide Starting Material

13.6 g (0.1 mol) of 1-methyl-1-benzyl-hydrazine (prepared from methylbenzyl amine and chloramine using standard methods [J. Chem. Ed. 485(1959)]) in 125 ml of toluene was cooled to 5° C. in an ice bath. Tothis was gradually added, with vigorous stirring over a one-hour period,a solution of 21.17 g (0.1 mol) of p-isopropylphenyl chloromethylanalide (prepared from chloracetyl chloride and p-isopropylphenyl amine)dissolved in 100 ml of toluene. Throughout the addition, the temperaturewas maintained at 5° C. The reaction mixture was then allowed to warm toroom temperature, and was stirred overnight. The precipitated solidhydrazinium salt was filtered, washed with cold toluene and dried in avacuum oven at 60° C./30″ to yield 34.3 g (98%) of racemic product. Thisracemate was slurried at room temperature overnight in 100 ml ethanoland a slight molar excess of moist silver oxide was added and themixture was stirred at room temperature overnight. The mixture was thenfiltered into an ethanolic solution containing an equivalent ofD-tartaric acid in the minimum amount of solvent. The alcoholic filtratewas concentrated to approximately 20% of its volume and diethylether wasadded until turbidity was observed. The turbid solution was then cooledat 0° C. overnight and the crystals were collected by filtration. Thesolid substance was purified by recrystallization from ethanol/ether toyield the desired pure diastereomeric salt, which was subsequentlyconverted to the iodide form by precipitation from a water-ethanolsolution of the tartrate (made alkaline by the addition of sodiumcarbonate) on treatment with an equivalent of solid potassium iodide,characterized by HPLC on Crownpack™ CR(+) chiral stationary phase(Daicell Chemical Industries Ltd.) using pH 2 aqueous mobile phase. NMR(DMSO-d₆): chemical shifts, peak integrations & D₂O exchange experimentsdiagnostic for structure.

14. EXAMPLE: Synthesis of a Peptide Mimetic Inhibiting Human Elastase

To 4.36 g (0.01 mol) of the chloromethylaminimide above in 50 ml ethanolwas added a solution of 1.86 g (0.01 mol) of1-methyl-1-isobutyl-2-N-trifluoroacetyl hydrazone (prepared from thereaction of trifluoroacetic anhydride with 1-methyl-1-isobutylhydrazine(from methyl isobutyl amine and chloramine) using standard acylationconditions) in 50 ml of ethanol. The mixture was heated to reflux,stirred at reflux for 4 hours, then cooled to room temperature andtitrated with 10% (w/v) KOH in methanol to the phenolphthalein endpoint.The mixture was filtered and the solvent was removed in vacuo on arotary evaporator. The residue was taken up in benzene and againfiltered. Removal of the benzene on the rotary evaporator yielded 5.7 g(95%) of the mixed (R)—(S) and (S)—(S) aminimide diastereomers. Thedesired (S)—(S) isomer was obtained pure by normal-phase chromatographicpurification over silica. This product is useful as a competitiveinhibitor for human elastase, characterized by HPLC on Crownpack™ CR(+)chiral stationary phase (Daicell Chemical Industries Ltd.) using pH 2aqueous mobile phase. NMR (DMSO-d₆): chemical shifts, peak integrations& D₂O exchange experiments diagnostic for structure.

Synthesis of the Chiral Chloroaminimide

A mixture of 4.87 g (0.01 mol) of the hydrazinium iodide enantiomerprepared as described in 5.2.3, 1.0 g (0.0106 mol) chloroacetic acid and1.24 g (0.011 mol) chloroacetyl chloride, contained in a micro reactionflask equipped with a drying tube, was heated at 105° C. for 1 hour withan oil bath. The (homogeneous) reaction mixture was then cooled to roomtemperature and extracted with 4×20 ml of ethyl ether to removechloracetyl chloride and chloracetic acid. The residual semisolid masswas dissolved in the minimum amount of methanol and titrated with 10%KOH in methanol to the phenolphthalein end point. The precipitated saltswere filtered and the filtrate was evaporated to dryness on a rotaryevaporator at 40° C. The residue was then taken up in benzene andfiltered. The solvent was removed on a rotary evaporator to give 3.88 g(89%) of the (S)—aminimide enantiomer, characterized by CDCL₃ NMRspectrum and D₂O exchange experiments and used directly in the next stepin the synthesis (see above).

Synthesis of the Chiral Aminimide

18.4 g (0.1 mol) of 1-(5′[3′-methyl uracil]methyl)-1-methylhydrazine(prepared by the alkylation of 2-methylphenylhydrazone with5-chloromethyl-3-methyl uracil in ethanol, as described in 24 J. Org.Chem. 660 (1959) and references cited therein, followed by removal ofthe benzoyl group by acid hydrolysis) in 100 ml toluene was cooled to 5°C. in an ice bath and a solution of 21.1 g (0.1 mol)p-isopropylphenyl-chloromethylanalide (prepared from chloracetylchloride and p-isopropylanaline), in 100 ml of toluene, was addedthereto with vigorous stirring over a 1-hour period, maintaining atemperature of 5° C. The reaction mixture was then allowed to warm toroom temperature and was stirred overnight. The solution was cooled to0° C. and the precipitated hydrazinium chloride salt was filtered,washed with cold toluene and dried in a vacuum oven at 40° C./30″ toyield 4.77 g (98%) of crude racemic product. This racemate was slurriedin 100 ml ethanol, a slight molar excess of moist silver oxide wasadded, and the mixture was stirred at room temperature overnight. Thisracemate was resolved via its tartrate salts and isolated as the iodideusing the method of Singh, above, characterized by HPLC on Crownpack™CR(+) chiral stationary phase (Daicell Chemical Industries Ltd.) usingpH 2 aqueous mobile phase. NMR (DMSO-d₆): chemical shifts, peakintegrations & D₂O exchange experiments diagnostic for structure.

Synthesis of 3-methyl-5-chloromethyluracil

A. 74.08 g (1 mol) of N-methyl urea and 216.2 g (1 mol)diethylethoxymethylenemalonate were heated together at 122° C. for 24hours, followed by 170° C. for 12 hours, to give the3-methyluracil-5-carboxylic acid ethyl ester in 35% yield (followingrecrystallization from ethyl acetate).

B. 30 g 3-methyluracil-5-carboxylic acid ethyl ester was saponified with10% NaOH to yield the free acid in 92% yield (after standard work-up andrecrystallization from ethyl acetate).

C. 20 g of 3-methyluracil-5-carboxylic acid was decarboxylated at 260°C. to give a quantitative yield of 3-methyluracil.

D. 3-methyluracil-5-carboxylic acid was treated with HCl and CH₂ usingstandard chloromethylation conditions to give 3-methyl-5-chloromethyluracil in 52% yield (following standard work-up and recrystallizationfrom ethyl acetate). NMR (DMSO-d₆): chemical shifts, peak integrations LD₂O exchange experiments diagnostic for structure.

13. EXAMPLE: Synthesis of a Peptide Mimetic Inhibiting the HIV Protease

This example teaches the synthesis of a competitive inhibitor for theHIV protease with enhanced stability, based on the insertion of a chiralaminimide residue into the scissile bond position of the substrateAc—L—Ser(Bzl)—L—Leu—L—Phe—L—Pro—L—Ile—L—Val—OMe (see, e.g., 33 J. Med.Chem. 1285 (1990) and references cited therein).

0.735 gms (1 mmol) of Ac—Ser(Bzl)—Leu—Asn—Phe——CO—NH—NC₅H₁₀ is dissolvedin the minimum amount of DMF, and 0.344 g of BrCH₂CONH—Val—Ile—OMe,prepared by treatment of H₂N—Val—Ile—OMe with (BrCH₂CO)₂O according tothe method of Kent (256 Science 221 (1992), is added thereto. Themixture is heated to 60° C. and stirred at this temperature overnight.At this point the DMF is removed under high vacuum, and the desired (S)isomer is obtained from the enantiomeric mixture after neutralization bystandard normal-phase silica chromatography to yield the protectedpeptide. The side chain blocking groups are subsequently removed usingstandard peptide deprotection techniques to yield the productAc—Ser—Leu—Asn—Phe—CON⁻N⁺(C₅H₁₀)—CH₂—CO—NH—Val—Ile—OMe, useful as aenhanced stability competitive inhibitor for the HIV protease.

Synthesis of the Tetrapeptide Hydrazone

0.653 g (1 mmol) of AcSer(Bzl)—Leu—Asn—Phe—OH, prepared via standardpeptide synthesis techniques (see 33 J. Med. Chem. 1285 (1990) andreferences cited therein), is coupled with 0.10 g (1 mmol) of1-aminopiperidine using standard peptide-coupling methods andchemistries (see 33 J. Org. Chem. 851 (1968)) to give a 97% yield of thehydrazide, isolated by removal of the reaction solvent in vacuo.

14. EXAMPLE: Preparation of Chiral Monomer Useful in PolymerizationYielding Crosslinked Polymer-Chains

3.18 g (0.01 mol) of (S)-1-methyl-1-ethyl-1-p-vinyl-benzylhydraziniumiodide, prepared from p-vinylbenzyl chloride and1-methyl-1-ethylhydrazine using standard alkylation conditions, andisolated as the (S)-enantiomer by the method of Singh (103 J. Chem. Soc.604 (1913)), were added to 75 ml of anhydrous t-butanol. The mixture wasstirred under nitrogen and 1.12 g (0.01 mol) of potassium t-butoxide wasadded. The mixture was stirred for 24 hours at room temperature and thereaction mixture was diluted with 75 ml of anhydrous THF, cooled in anice bath and 1.39 g (0.01 mol) of 2-vinyl-4,4-dimethylazlactone in 50 mlof THF were then added over a 15-min. period. When addition wascomplete, the mixture was allowed to warm to room temperature andstirred at room temperature for 6 hours. The solvent was stripped underaspirator vacuum on a rotary evaporator to yield 3.0 g (92%) of crudemonomer. The product was recrystallized from ethyl acetone at −30° C. toyield pure crystalline momomer, useful for fabricating crosslinkedchiral gels, beads, membranes and composites for chiral separations,particularly for operation at high pH. NMR (CDCl₃) chemical shifts,presence of vinyl groups in 6 ppm region, vinyl splitting patterns, peakintegrations and D₂O experiments diagnostic for structure. FTIR absenceof azlactone CO band in 1820 cm⁻¹ region.

15. EXAMPLE: Functionalization of Silica with an Oxazolone Followed byConversion to a Chiral Aminimide Useful in the Resolution of RacemicCarboxylic Acids

2.81 g (0.01 mol) of (S)-1-methyl-1-ethyl-1phenyl-hydrazinium iodide,prepared by the method of Singh (103 J. Chem Soc. 604 (1913)), was addedto 100 ml anhydrous t-butanol. The mixture was stirred under nitrogenand 1.12 g (0.01 mol) potassium t-butoxide was added. The mixture wasstirred for 24 hours at room temperature, after which the reactionmixture was diluted with 100 ml anhydrous THF. To this mixture was added5.0 g silica functionalized with the Michael-addition product of(S)-4-ethyl-4-benzyl-2-vinyl-5-oxazolone to mercaptopropyl-functionalsilica. This mixture was stirred at room temperature for 8 hours. Thefunctionalized silica was collected by filtration and successivelyreslurried and refiltered using 100-ml portions of toluene (twice),methanol (four times) and water (twice). The resulting wet cake wasdried in a vacuum oven at 60° C. under 30″ vacuum to constant weight,yielding 4.98 g of chiral-aminimide-functionalized silica, useful forthe separation of racemic mixtures of carboxylic acids, such asibuprofen, ketoprofen and the like.

16. EXAMPLE: Functionalization of Silica With a Chiral Aminimide for usein the Separation of Mandelates

10.0 g epoxy silica (15μ Exsil C-200 silica) was slurried in 75 mlmethanol and shaken to uniformly wet the surface. To this slurry wasadded 6.01 g (0.01 mol) 1,1-dimethylhydrazine, and the mixture wasallowed to stand at room temperature with periodic shaking for 45 min.32.5 g (0.1 mol) of (S)-3,5-dinitrobenzoylvaline methyl ester was addedand the mixture was allowed to stand at room temperature with periodicshaking for three days. The functionalized silica was then collected byfiltration, re-slurried in 100 ml methanol and re-filtered a total offive times, then dried in a vacuum oven at 60° C./30″ overnight to give9.68 g of the product. This functionalized silica was slurry packed frommethanol into a 0.46×15 cm stainless steel column and used to separatemixtures of mandelic acid derivatives under standard conditions.

Preparation of Epoxy Silica

50 g of 5μ C-200 Exsil silica (SA 250μ²/g) was added to 650 ml toluenein a two-liter three-necked round-bottomed flask equipped with a Teflonpaddle stirrer, a thermometer and a vertical condenser set up with aDean-Stark trap through a claisen adaptor. The slurry was stirred,heated to a bath temperature of 140° C. and the water was azeotropicallyremoved by distillation and collection in the Dean-Stark trap. The lossin toluene volume was measured and compensated for by the addition ofincremental dry toluene. 200 g of glycidoxypropyl trimethoxysilane wasadded carefully through a funnel and the mixture was stirred andrefluxed overnight with the bath temperature set at 140° C. The reactionmixture was then cooled to about 40° C. The resulting functionalizedsilica was collected on a Buechner filter, washed twice with 50 mltoluene, sucked dry, reslurried in 500 ml toluene, refiltered,reslurried in 500 ml methanol and refiltered a total of four times. Theresulting methanol wet cake was dried overnight in a vacuum oven set for30″ at 60° C. to yield 48.5 g of epoxy silica.

Synthesis of N-3,5-Dinitrobenzoyl-(S)-Valine Methyl Ester

13.12 g (0.1 mol) of (S)-valine methyl ester was added with stirring toa solution of 8 g (0.2 mol) sodium hydroxide in 50 ml of water, cooledto about 10° C., and the mixture stirred at this temperature untilcomplete solubilization was achieved. 23.1 g (0.1 mol) of3,5-dinitrobenzoylchloride was then added dropwise with stirring,keeping the temperature at 10-15° C. with external cooling. After theaddition was complete, stirring was continued for 30 min. To thissolution was added over a 10-min. period 10.3 ml (1.25 mol) ofconcentrated hydrochloric acid, again keeping the temperature at 15° C.After this addition was complete, the reaction mixture was stirred foran additional 30 min. and cooled to 0° C. The solid product wascollected by filtration, washed well with ice water and pressed firmlywith a rubber dam. The resulting wet cake was recrystallized fromethanol/water and dried in a vacuum oven under 30″ vacuum at 60° C. toyield 28.5 g (90%) of N-3,5-dinitrobenzoyl-(S)-valine methyl ester. NMR(CDCl₃): chemical shifts, splitting patterns, integrations and D₂Oexchange experiments diagnostic for structure.

17. EXAMPLE: Preparation of Aminimide-Containing Ion-Exchange SilicaMatrix

This example describes preparation of an aminimide-functionalizedion-exchange silica matrix using epoxy silica as the support to bemodified. The reaction sequence is:

25 g of epoxy silica (15μ Exsil AWP 300 silica, with surface area of 100m²/g) was slurried in 100 ml methanol until completely wetted by thesolvent. 10.2 g of 1,1-dimethylhydrazine were then added with swirlingand the mixture allowed to stand at room temperature for 3 hours. 24.7 gof Et₂NCH₂CH₂COOEt were then added and the mixture kept at roomtemperature with periodic shaking for 2 days.

The diethylaminoethyl (DEAE) functionalized silica was collected byfiltration, re-slurried in 100 ml methanol and re-filtered a total offive times. The packing was dried in a vacuum oven at 60° C./30″overnight. A 1.0 ml bed of this material was then packed in a 15 mM NaAcbuffer at pH 7.7. The column was then equilibrated with 15 mM NaAcbuffer at pH 5.6, and a solution of 1 mg/ml ovalbumin in this buffer runthrough the bed at a flow rate of 1.6 ml/min. A total of 59.2 ml ofprotein solution was run.

The column was then washed with 41.7 ml of 15 mM NaAc buffer at pH 5.58and at a flow rate of 3.9 ml/min. The bound protein was eluted using23.4 ml of 0.5M NaCl at a flow rate of 3.9 ml/min. The eluent (15.2 ml)was then collected and the transmission of an aliquot measured at 280 mμwith a spectrophotometer. The ovalbumin concentration was determinedfrom a calibration curve. The total amount of ovalbumin collected was63.7 mg.

18. EXAMPLE: Preparation of Aminimide-Containing Size-Exclusion SilicaMatrix

This example describes preparation of an aminimide-functionalizedsize-exclusion silica matrix using the epoxy silica support describedabove.

10.0 g of epoxy silica (15μ Exsil C-200 silica, with surface area of 250m²/g) was slurried in 75 ml of methanol and shaken to uniformly wet thesurface. To this slurry was added 10.2 g of 1,1-dimethylhydrazine. Themixture was allowed to stand at room temperature with periodic shakingfor 45 min.

15 g of ethyl acetate were then added and the mixture allowed to standat room temperature with periodic shaking for 3 days. The functionalizedsilica was then collected by filtration, re-slurried in 100 ml methanol,re-filtered a total of five times and dried in a vacuum oven at 60°C./30″ overnight. The functionalized silica was slurry packed frommethanol into a 10 mm interior-diameter jacketed glass column withadjustable pistons to provide an 8 cm-long packed bed. This packing wasused to separate mixtures of polyethylene glycol polymers of varyingmolecular weight with good resolution using a mobile phase.

In a second experiment, the bulk packing was found to selectively adsorbpolyethylene-glycol functionalized hemoglobin from serum samples takenfrom test animals that had been treated with this derivative as a bloodsubstitute. Filtration of the serum, after treatment with the bulkpacking, gave a serum free from the functionalized hemoglobin, thusallowing blood screening or testing by means of standard methods.

19. EXAMPLE: Coating of a Silica Matrix with Hydroxypropyl CelluloseFunctionalized with an Aminimide

Hydroxypropylcellulose is mono-functionalized by reaction, under strongalkaline conditions (preferably provided by a strong base, such aspotassium t-butoxide) with ClCH₂CON⁻N⁺(CH₃)₃. The result is replacementof approximately one hydroxyl group in each saccharide unit with theaminimide as follows:

The resulting aminimide derivative is coated onto a surface (e.g.,silica). Upon heating to 140° C., the N(CH₃)₃ group leaves, resulting information of an isocyanate moiety:

The isocyanate groups then react with unreacted hydroxyl groups on thesaccharide units to produce a cross-linked coating.

Alternatively, the cellulose can be coated onto the surface andimmobilized using standard techniques (e.g., reaction with bisoxiranes),and then mono, di- or tri-substituted with desired aminimide derivativesas described above.

The foregoing reaction sequence can also be employed with polymers oroligomers bearing NH or SH groups instead of hydroxyl groups and canalso be utilized to fabricate structures such as crosslinked cellulosemembranes.

20. EXAMPLE: Coating of a Silica Matrix via Polymerization of anAminimide on the Matrix

This example illustrates an alternative immobilization technique,namely, polymerizing aminimide recursors containing vinyl groups andwhich have been coated onto a surface. The chemistry resembles theapproach described above, except polymerization forms a sturdy shellaround an existing support rather than creating a solid block ofmaterial.

This sequence makes use of the reaction described above. An epoxide,

is combined with methyl methacrylate and dimethylhydrazine as set forthin 2.a above to form CH═C(CH₃)—CO—NN(CH₃)₂—CH₂—CH(OH)—CH₂—N⁺(CH₃)₃Cl⁻.3.11 g of this material and 0.598 g n-methylol acrylamide were dissolvedin 75 ml of methanol, and 3.54 ml of water were then added. To thissolution were added 15 g of epoxy silica (15μ Exsil AWP 300 silica, withsurface area of 100 m²/g).

The mixture was stirred in a rotary at room temperature for 15 min andthen stripped using a bath temperature of 44° C. to a volatiles contentof 15% as measured by weight loss (from 25-200° C. with a sun gun). Thecoated silica was slurried in 100 ml of isooctane containing 86 mg ofVAZO-64 dissolved in 1.5 ml toluene which had been de-aerated withnitrogen. The slurry was thoroughly de-aerated with nitrogen and thenstirred at 70° C. for two hours.

The coated silica was collected by filtration and washed three times in100 ml methanol and air dried. The silica was heated at 120° C. for 2hours to cure the coating. 13.1 g of coated silica were obtained. A 1 mlbed of this material was packed in an adjustable glass column andsuccessfully used to separate BSA from lactoglobulin.

21. EXAMPLE: Preparation of Silica Support Containing CrosslinkedAminimide Polymer Chains

In this example, an epoxy-functionalized surface is reacted withdisubstituted hydrazine, a bisepoxide and a triester to form acrosslinked network of aminimide chains attached covalently to thesurface as follows:

The reaction can be carried out in water at room temperature withoutspecial conditions.

22. EXAMPLE: Preparation of Cross-Linked Porous Aminimide Ion-ExchangeBeads

This example describes preparation of three-dimensional cross-linkedporous copolymeric aminimide ion-exchange beads. It involves reaction ofthree monomers:

Monomer A: CH₂═CH—CON—N⁺(CH₃)₃

Monomer B: CH₂═C(CH₃)—CON⁻N⁺(CH₃)₂—CH₂—CH(OH)—CH₂—N⁺(CH₃)₃Cl⁻

Crosslinker: CH₂═CH—CO—NH—C(CH₃)₂—CON⁻N⁺(CH₃)₂—CH₂—Ph—CH═CH₂

where Ph is phenyl.

Preparation of Monomer A: This monomer was prepared according to themethod described in 21 J. Polymer Sci., Polymer Chem. Ed. 1159 (1983).

Preparation of Monomer B: 30.3 g (0.2 mol) of glycidyl-trimethylammoniumchloride was dissolved in 100 ml of methanol and filtered free ofinsolubles. 22 g (0.22 mol) of methyl methacrylate was added thereto,followed by 12 g (0.2 mol) of 1,1-dimethylhydrazine. The solution grewwarm and turned slightly pink. It was allowed to stand for 6 days atroom temperature, and was then treated with charcoal, filtered, andconcentrated on a rotary evaporator at 55° C. and 10 mm to produce athick lavendar-colored, viscous material. This material was trituratedwith diethylether and hot benzene and dissolved in the minimum amount ofmethanol. The mixture was then treated with charcoal, filtered, heatedto boiling and brought to the cloud point with ethyl acetate. Theresulting solution was allowed to stand at 0° C. for a week. The whitecrystals that formed were collected by filtration, washed with coldethyl acetate and dried in a vacuum oven at room temperature to yield7.3 g of monomer B.

Preparation of Monomer C: 18 g (0.3 mol) of 1,1-dimethylhydrazine wasdissolved in 50 ml CH₂Cl₂ and cooled in an ice bath with stirring. 41.7g (0.3 mol) of vinylazlactone in So ml CH₂Cl₂ were added slowly to keepthe temperature below 5° C. The clear solution was stirred and allowedto come to room temperature over 1 hour (resulting in formation of awhite solid) and was stirred at room temperature for an additional 1.5hours. The white solid was collected by filtration, re-slurried in 100ml CH₂Cl₂ and re-filtered. It was then dried in a vacuum oven at roomtemperature overnight to yield a total of 26.81 g of the intermediateCH₂═CH—CO—NH—C(CH₃)₂—CO—NH—N—(CH₃)₂. 10.0 g (0.05 mol) of thisintermediate and 7.66 g (0.05 mol) of vinyl benzyl chloride weredissolved in a mixture of 50 ml ethanol and 50 ml CH₃CN. The solutionwas refluxed for 4 hours under a nitrogen stream. It was then cooled toroom temperature and concentrated on a rotary evaporator at 55° C. toproduce a thick yellow oil. The oil was triturated three times withdiethylether to yield 17.08 g of an off-white solid. This solid wasdissolved in 100 ml of hot methanol and filtered through a celite pad toremove a small amount of gelatinous material, and the clear filtrate wasstripped to yield 10.0 g of Monomer C as a white solid.

Polymerization: 1 ml of the emulsifier Span 80 and 175 ml of mineral oilwere introduced into a 500 ml round-bottomed flask equipped with stirrerand a heating bath. The mixture was mechanically stirred at 70 RPM andbrought to a temperature of 55° C. 40.5 g of monomer A, 7.2 g of monomerB and 5.7 g of the cross-linker were dissolved in 100 ml ofdemineralized water and heated to 55° C. To this solution were added 150mg of ammonium persulfate, and the mixture was then poured into thestirred mineral oil. The agitation was adjusted to produce a stableemulsion with an average droplet diameter of approximately 75μ (asdetermined with an optical microscope).

After 15 min, 0.15 ml of TMED was added and stirring was continued foran additional 45 min. The reaction mixture was cooled and allowed tostand overnight. The supernatant mineral oil phase was removed byaspiration and the beads were collected by decantation. The beads werewashed three times with a 0.05% solution of Triton X-100 indemineralized water to remove any remaining mineral oil and then washedwith water and allowed to settle. The water was removed by decantation.

This procedure was repeated a total of five times. The beads obtained atthe conclusion of the foregoing steps had a mean diameter ofapproximately 75μ and an ion-exchange capacity of 175 μeq/ml.

23. EXAMPLE: Preparation of an Aminimide-Based Electrophoretic Gel

This example describes preparation of an aminimide electrophoresis gel.As a control, the standard Sigma protein electrophoresis mix (availablefrom Sigma Chemical Co., St. Louis, Mo.) was run on anacrylamide/methylene bisacrylamide linear gradient gel prepared using agradient maker with 5% and 12.5% monomer solutions, as shown below. Thegel was overlayed with isobutanol and allowed to polymerize overnight.

Monomer 5% Monomer 12.5% Lower Tris 5.0 ml 5.0 ml H₂O 11.7 ml 4.7 ml 30%Acrylamide 3.3 ml 8.3 ml Glycerol — 2.0 ml Ammonium Persulfate 30 μl 30μl TMED 15 μl 15 μl

Lower Tris 1.5M: 6.06 g Tris base, 8 ml 10% SDS, volume adjusted to 90ml with double-distilled water. The pH was adjusted to 6.0 withconcentrated HCl, and the final volume adjusted to 100 ml with DD water.

Acrylamide 30% w/v: 29.2 g acrylamide, 0.8 g of methylene bisacrylamideand 100 ml DD water.

SDS 10% w/v: 10 g of SDS dissolved in DD water and adjusted to a volumeof 100 ml.

Ammonium persulfate 10%: 0.1 g ammonium persulfate was dissolved in 0.9ml DD water. The solution was used within 4 hours of preparation.

TMED: used directly as obtained from Sigma Chemical Co., St. Louis, Mo.,under the tradename TMEDA.

A second gel was prepared by replacing the acrylamide with an equalweight of the aminimide monomer CH₂═CH—CO—N—N(CH₃)₃ and the proteinstandard was run in the same way as the first.

Separation of proteins with the aminimide gel were equivalent to theacrylamide gel, but the aminimide gel produced Rf (i.e., the ratio ofdistance traversed by a particular protein to the distance traversed bythe solvent front) levels approximately 20% higher than those of theacrylamide gel.

24. EXAMPLE: Preparation of Aminimide-Based Latex Particles

This example describes preparation of latex particles containing anaminimide comonomer.

591.1 ml of distilled water was charged to a three-necked round-bottomedflask. A nitrogen dip tube was placed below the liquid level and thenitrogen flow rate set to 2 cm³/min. The solution was mechanicallyagitated with a Teflon paddle at 250 RPM and heated to 80° C. over ahalf-hour period. In a separate flask were dissolved 121.6 g of butylacrylate, 54.6 g of ethyl acrylate, 13.0 g of acrylic acid, 9.97 g ofmethyl methacrylate, 59.7 g of the aminimide monomerCH₂═CH—CO—N—N(CH₃)₂—CH₂—CH₂—OH and 0.92 g of Aerosol TR-70 so as toobtain solution without exceeding a temperature of 25° C. Whencompletely dissolved, 1.53 g of additional TR-70 were added and themixture was then stirred until solution was achieved.

20.7 ml of distilled water was purged with nitrogen for 10 min and 1.59g of K₂S₂O, is dissolved in it. This persulfate solution was added tothe heated water in the reaction flask after it stabilized at 80° C. Thenitrogen dip tube was raised and a nitrogen blanket was maintained. Themonomer mix was pumped in at a steady, calibrated rate such that theconstant addition took exactly 4 hours. When addition was complete, thelatex was post-heated at 80° C. for 1 hour, cooled to 25° C. andtitrated to pH 5.0 by dropwise addition of triethylamine (approximately20 cm³) over 20 min with agitation. The latex was then filtered throughcheese cloth and stored. Average particle size was measured at about0.14μ.

It should be apparent to those skilled in the art that othercompositions and processes for preparing the compositions notspecifically disclosed in the instant specification are, nevertheless,contemplated thereby. Such other compositions and processes areconsidered to be within the scope and spirit of the present invention.Hence, the invention should not be limited by the description of thespecific embodiments disclosed herein but only by the following claims.

What is claimed is:
 1. A composition having the structure

wherein a. A and B are the same or different, and each is a chemicalbond; hydrogen; an electrophilic group; a nucleophilic group; R; anamino acid derivative; a nucleotide derivative; a carbohydratederivative; an organic structural motif; a reporter element; an organicmoiety containing a polymerizable group; or a macromolecular component,wherein A and B are optionally connected to each other or to otherstructures and R is as defined below; b. X and Y are the same ordifferent and each represents a chemical bond or one or more atoms ofcarbon, nitrogen, sulfur, oxygen or combinations thereof; c. R and R′are the same or different and each is an alkyl, cycloalkyl, aryl,aralkyl or alkaryl group or a substituted or heterocyclic derivativethereof, wherein R and R′ may be different in adjacent n units and havea selected stereochemical arrangement about the nitrogen atom to whichthey are attached; d. G is a chemical bond or a connecting group thatincludes a terminal carbon atom for attachment to the quaternarynitrogen and G may be different in adjacent n units; and e. n≧1;provided that, (1) if G is a chemical bond, Y includes a terminal carbonatom for attachment to the quaternary nitrogen; and (2) if n is 1, X andY are chemical bonds and R and R′ are the same, A and B are differentand one is other than H or R.
 2. The composition of claim 1 wherein n>2.3. The composition of claim 1 wherein at least one of R and R′ includesa hydroxyl containing substituent.
 4. The composition of claim 1 whereinG includes at least one of an aromatic ring, a heterocyclic ring, acarbocyclic moiety, an alkyl group or a substituted derivative thereof.5. The composition of claim 1 wherein A and B are the same.
 6. Thecomposition of claim 1 where R and R′ are different so that thecomposition is chiral.
 7. The composition of claim 1 wherein at leastone of A and B is a terminal-structural moiety of formula T-U, wherein:a. U is selected from the group consisting of aliphatic chains havingfrom 2 to 6 carbon atoms, substituted or unsubstituted aryl, substitutedor unsubstituted cycloalkyl, and substituted or unsubstitutedheterocyclic rings; and b. T is selected from the group consisting of—OH, —NH₂, —SH, (CH₃)₃N⁺—, SO₃—, —COO⁻, CH₃, H and phenyl.
 8. Thecomposition of claim 1 wherein at least one of A and B isHO—CH₂—(CHOH)_(n)—.
 9. The composition of claim 1 wherein A and B arepart of the same cyclic moiety.
 10. The composition of claim 1, whereinA and B are the same or different, and each is selected from an aminoacid derivative, a nucleotide derivative and a carbohydrate derivative.11. The composition of claim 1, wherein one of A and B is amacromolecular component, and the other is selected from an amino acidderivative, a nucleotide derivative and a carbohydrate derivative. 12.The composition of claim 1, wherein one of A and B is an organic moietycontaining a polymerizable group, and the other is selected from anamino acid derivative, a nucleotide derivative and a carbohydratederivative.
 13. The composition of claim 1, wherein A and B are the sameor different, and each is selected from hydrogen and R.
 14. Thecomposition of claim 1, wherein A and B are the same or different, andeach is selected from an electrophilic group and a nucleophilic group.